Dalbavancin compositions for treatment of bacterial infections

ABSTRACT

The invention provides methods and compositions for treatment of bacterial infections. The composition may be a combination of factors, which include A 0 , A 1 , B 1 , B 2 , C 0 , C 1 , isoB 0 , and MAG, in the presence of low level solvent. Methods of the invention include administration of dalbavancin formulations for treatment of a bacterial infection, in particular a Gram-positive bacterial infection of skin and soft tissue. Dosing regimens include multiple dose administration of dalbavancin, which often remains at therapeutic levels in the bloodstream for at least one week, providing prolonged therapeutic action against a bacterial infection. Dosing regimens for renal patients are also included.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.10/834,395, filed Apr. 27, 2004, which is a continuation-in-part of U.S.application Ser. No. 10/714,261, filed Nov. 14, 2003, which claims thebenefit of U.S. Provisional Patent Application Ser. Nos. 60/427,654,filed Nov. 18, 2002, 60/485,694, filed Jul. 8, 2003, 60/495,048, filedAug. 13, 2003, and 60/496,483, filed Aug. 19, 2003, the disclosures ofall of which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

This application relates to dalbavancin compositions and methods of useof such compositions in methods of treatment of bacterial infections.

BACKGROUND OF THE INVENTION

According to the U.S. Center for Disease Control and Prevention,nosocomial bloodstream infections are a leading cause of death in theUnited States. Approximately five percent of the seven million centralvenous catheters (CVCs) inserted annually in the United States areassociated with at least one episode of bloodstream infection(approximately 350,000 a year). Catheter-related bloodstream infectionsoccur when bacteria enter the bloodstream through an intravenouscatheter and can be life threatening.

Skin and soft tissue infections (SSTIs) (also known as complicatedand/or uncomplicated skin and skin structure infections (SSSIs)) are acommon medical condition and often the consequence of trauma or surgicalprocedures. Staphylococcus aureus and Streptococcus pyogenes are thepathogens most frequently isolated from patients with deep tissueinfections, although any pathogenic organism, including those found onhealthy skin, may cause infection. Many SSTIs are mild to moderate inseverity, permitting successful treatment with oral antimicrobial agentsand local cleansing. In contrast, more severe or complicated infections,which frequently occur in patients with underlying risk factors (e.g.,vascular compromise, diabetes) and/or infections caused bydifficult-to-treat or multiply-resistant bacteria, may require potentintravenous antimicrobial therapy and aggressive surgical debridement.

Staphylococci are a clinical and therapeutic problem and have beenincreasingly associated with nosocomial infections since the early1960s. The coagulase-positive species methicillin-resistantStaphylococcus aureus (MRSA) has long been problematic in bothcommunity-acquired and nosocomial infections, and severalcoagulase-negative staphylococci have been recognized as opportunistichuman pathogens, especially in the treatment of critically ill patientsin intensive care units. Another major cause for clinical concern is theincreasing isolation of penicillin-resistant Streptococcus pneumoniaestrains in many parts of the world.

The glycopeptide antibiotics vancomycin and teicoplanin have been usedagainst serious nosocomial infections caused by multi-drug-resistantGram-positive pathogens, particularly MRSA, coagulase-negativestaphylococci (CoNS), and enterococci. Vancomycin and teicoplanin areused for infections caused by MRSA, and until recently, all isolateswere uniformly susceptible. However, the isolation of Staphylococcusaureus strains with intermediate susceptibility or resistance toteicoplanin as well as vancomycin has now been reported with increasingfrequency. A number of vancomycin-resistant strains, classified “VanA,”“VanB,” or “VanC,” based on the mechanism of resistance, have beenreported. Thus, alternative treatment options are needed.

Teicoplanin is at least as active as vancomycin against mostGram-positive bacteria and appears to cause fewer adverse events. Bothforms of treatment require at least once daily dosing to effect completerecovery. Currently, the therapeutic options for severe infectionscaused by some of these pathogens are quite limited. The emergingresistance of Gram-positive pathogens to vancomycin makes theavailability of new antibiotics with potential for increasedeffectiveness highly desirable.

In addition, less frequent dosing regimens than currently-availabletherapies would be desirable to enhance patient comfort, especially forparenteral, e.g., intravenous or intramuscular, antibioticadministration. Hospital stays are sometimes necessitated by the needfor multi-daily antibiotic administration by parenteral means, and lessfrequent dosing would be advantageous to permit such treatment to bedone on an outpatient basis.

Although less frequent dosing is a desirable feature of an antibioticadministration regimen, the “pharmaceutical window,” i.e., the toxicityprofile, of the administered antibiotic must be sufficiently acceptableto permit a large single dose to be administered without jeopardizingtreatment by causing severe adverse reactions in the treated patient.Further, even when an antibiotic exhibits a suitable pharmaceuticalwindow, less frequent dosing is possible only if the antibiotic exhibitsa suitable serum half-life to maintain therapeutic effectiveness overthe dosing interval desired. The serum half-life of an antibioticdictates both the longevity of a drug in vivo and the length of timeafter administration when the serum level will reach a minimum troughlevel which is still bactericidally effective. The serum trough levelover time after administration of a first dose of antibiotic dictateswhen a further dose must be administered to retain a minimumbactericidal level of the antibiotic in vivo.

Recently, successful glycopeptide antibiotics have been rationallysynthesized from natural glycopeptides. For example, the semisyntheticglycopeptide dalbavancin was synthesized from the natural antibiotic A40926, originally isolated from an Actinomadura culture (Malabarba etal., 1998, U.S. Pat. No. 5,750,509). Dalbavancin has shown greaterefficacy against various bacterial strains than vancomycin or theantibiotic linezolid and represents a promising new treatment for skinand soft tissue infections (see, e.g., Jabés et al., 2004, Antimicrob.Agents Chemother. 48:1118-1123). According to U.S. Pat. No. 5,750,509,dalbavancin is a glycopeptide antibiotic with a monomethyl moiety at itsN¹⁵ amino (see FIG. 1 for numbering), and this N¹⁵-monomethyl aminocould be free (i.e. —NHCH₃) or protected with an amino protecting groupsuch as t-butoxycarbonyl, carbobenzyloxy, arylalkyl or benzyl. Themethod for making certain of the dalbavancin components reported in the'509 patent also produced N¹⁵,N¹⁵-dialkyl analogs of dalbavancin inminor-quantities, but these molecules were not characterized.

In view of the above pathogens, further antibiotics possessing activityagainst one or more microbes, including antibiotic resistant bacteria,would be of commercial value and would satisfy a long-felt need in theart.

SUMMARY OF THE INVENTION

The invention provides compositions, methods and kits for treatment orprevention of a bacterial infection with dalbavancin. Surprisingly,stabilized formulations of dalbavancin have been found to exhibit both apharmaceutical window as well as a prolonged serum half-life to permittreatment regimens of about once every 5-7 days or longer, whileretaining antibacterial properties in vivo.

Accordingly, in one aspect, a pharmaceutical composition is providedthat includes a unit dose of dalbavancin in an amount sufficient toprovide a therapeutically or prophylactically effective plasma level ofdalbavancin in an individual for at least five days, a stabilizer, and apharmaceutically acceptable carrier.

Pharmaceutical compositions of the invention are generally formulated ina pharmaceutically acceptable form for administration to an individual,such as a pharmaceutically acceptable aqueous formulation. Suchpharmaceutical compositions are preferably administered by parenteral,e.g., intravenous or intramuscular, routes. Accordingly, in thispreferred embodiment, these pharmaceutical compositions are typicallysterile.

In some embodiments, a unit dose of dalbavancin is provided in drypowder (e.g., lyophilized) form and reconstituted in a pharmaceuticallyacceptable carrier, such as a sterile aqueous formulation, prior toadministration to an individual. In one embodiment, the pharmaceuticallyacceptable carrier includes 5% dextrose in water. A pharmaceuticalcomposition of the invention may be administered to a mammal in need oftreatment or prevention of a bacterial infection, such as a human. Insome embodiments, a pharmaceutical composition may include at least oneantibiotic that is not dalbavancin, such as an antibiotic that iseffective (e.g., bactericidal) against a Gram-negative bacterium and/oran antibiotic that is effective against Gram-positive species againstwhich dalbavancin is not effective, such as VanA vancomycin-resistantbacterial strains.

The invention provides compositions, methods of making, and methods fortreatment or prevention of a bacterial infection with a room-temperaturestable dalbavancin pharmaceutical composition.

In some embodiments, one or more stabilizing substances are employed toinhibit degradation of one or more dalbavancin components during storageas a dry powder (e.g., lyophilized) formulation and/or as an aqueousformulation prior to administration to an individual. Over time,degradation can result in the undesirable formation of less activeand/or inactive components which could potentially cause adverse effectsin vivo. Preferred stabilizers include nonionic components such assugars or sugar alcohols, e.g., a mono-, di-, or polysaccharide, orderivative thereof, such as, for example, mannitol, lactose, sucrose,sorbitol, glycerol, cellulose, trehalose, maltose, or dextrose, ormixtures thereof.

In one embodiment, the invention encompasses a pharmaceuticalcomposition comprising stable dalbavancin.

In another embodiment, the invention encompasses a pharmaceuticalcomposition comprising dalbavancin and a stabilizer.

In yet another embodiment, the invention encompasses a pharmaceuticalcomposition comprising dalbavancin and a stabilizer at a pH of about1-7, more preferably 2-6. In another embodiment, the composition is at apH of about 3-5. The stabilizer may comprise a carbohydrate or an aminoacid. The carbohydrate may be mannitol, lactose, or a combination ofmannitol and lactose. The mannitol and lactose may be added in equal orunequal amounts. In one embodiment, equal amounts of mannitol andlactose are added and the pH is adjusted to about pH 4.5.

In another embodiment, the invention also encompasses a pharmaceuticalcomposition comprising dalbavancin and mannitol at a pH of about 3. Inone embodiment, the pH of the composition is about 3.3. In anotherembodiment, this composition may further comprise lactose. The lactoseand mannitol may be added in equal or unequal amounts.

In another embodiment, the invention also encompasses a pharmaceuticalcomposition comprising dalbavancin and a stabilizer, wherein thestabilizer comprises mannitol and lactose. The mannitol and lactose maybe added in equal amounts. The pH of this composition may optionallyrange from 1-7, more preferably 2-6, more preferably 3-5, morepreferably 4-5, more preferably approximately 4.5.

Glycopeptides, and dalbavancin in particular, are very unstable due tothe glycosidic linkage. There may be some degradation at roomtemperature and more degradation at 40° C. Some of the formulationsdescribed above may need special storage conditions. In particular,refrigeration may be desirable (e.g., −40 to 10° C., alternatively −20to 9° C., more preferably 2 to 8° C.). The formulations may additionallybe sterilized. They will form a stable, clear, particle free solutionwhen administered. The solution should be stable and not contain aprecipitate.

The pharmaceutical compositions described above preferably degrade by nomore than about 4% at about 25° C. after about 2 years, more preferablyby no more than about 3%, more preferably by no more than about 2%, morepreferably by no more than about 1%, more preferably by no more thanabout 0.5%. Alternatively, the pharmaceutical compositions describedabove have no more than about 4% MAG at about 25° C. after about 2years, more preferably have no more than about 3% MAG, more preferablyhave no more than about 2% MAG, more preferably have no more than about1% MAG, more preferably have no more than about 0.5% MAG.

In another embodiment, the pharmaceutical compositions described abovepreferably degrade by no more than about 6% at about 40° C. after about3-6 months, more preferably by no more than about 5%, preferably by nomore than about 4%, preferably by no more than about 3%, preferably byno more than about 2%, preferably by no more than about 1%.Alternatively, the pharmaceutical compositions described abovepreferably have no more than about 6% MAG at about 40° C. after about3-6 months, more preferably have no more than about 5% MAG, preferablyhave no more than about 4% MAG, preferably have no more than about 3%MAG, preferably have no more than about 2% MAG, and even more preferablyhave no more than about 1% MAG. A stable compound at 40° C. would bedesirable, especially in places that are not able to store the compoundsin a refrigerator or at room temperature (e.g., third world countriesand Indian reservations).

In yet another embodiment, the pharmaceutical compositions degrade by nomore than 3% at about 2-8° C. after about 2 years, more preferably by nomore than about 2%; more preferably by no more than about 1%; morepreferably by no more than about 0.5%. Alternatively, the pharmaceuticalcompositions have no more than 3% MAG at about 2-8° C. after about 2years, more preferably have no more than about 2% MAG; more preferablyhave no more than about 1% MAG; more preferably have no more than about0.5% MAG.

The invention includes dalbavancin compositions that may comprise acombination of any of the dalbavancin factors. These factors includedalbavancin factor A₀, A₁, B₁, B₂, C₀, C₁, isoB₀, and MAG.

The invention also include a drying process for reducing the level ofsolvent in the dalbavancin composition. The method includes the steps ofproviding wet dalbavancin comprising dalbavancin, water, and a solvent,drying the wet dalbavancin at a temperature of about 30° C. or less andat a vacuum pressure of about 50 mbar or less, until the water level ofthe wet dalbavancin is less than about 20% (w/w). Water is then added tothe wet dalbavancin and the drying step is repeated. These steps ofadding water and drying the wet dalbavancin are repeated until thesolvent level is less than about 3.0% (w/w). These steps can be repeatedonce, twice, three times, four times, five times, six times, or as manyas necessary to reach the desired solvent content. Similarly, theinvention also includes a dalbavancin composition that has a low levelof solvent. In one embodiment, the level of solvent may be below 3.0%(w/w), alternatively below 2.5% (w/w), alternatively below 2.0% (w/w),alternatively below 1.5% (w/w), alternatively below 1.0% (w/w),alternatively below 0.5% (w/w), alternatively below 0.1% (w/w). Thesolvent may be acetone. Alternatively, the solvent may be any ofethanol, methanol, propanol, butanol, ether, methylene chloride,tetrahydrofuran, chloroform, 1,4-dioxane, trichloroethylene, benzene,carbon tetrachloride, 1,2-dichloroethane, 1,1-dichloroethane,1,1,1-trichloroethane, acetonitrile, chlorobenzene, cyclohexane,dichloromethane, 1,2-dimethoxyethane, N,N-dimethylacetamide,N,N-dimethylformamide, 1,4-dioxane, 2-ethoxyethanol, ethyleneglycol,formamide, hexane, 2-methoxyethanol, methylbutyl ketone,methylcyclohexane, N-methylpyrrolidone, nitromethane, pyridine,sulfolane, tetralin, toluene, 1,1,2-trichloroethane, xylene, aceticacid, anisole, 1-butanol, 2-butanol, butyl acetate, tert-butylmethylether, cumene, dimethyl sulfoxide, ethyl acetate, ethyl ether, ethylformate, formic acid, heptane, isobutyl acetate, isopropyl acetate,3-methyl-1-butanol, methylethyl ketone, methylisobutyl ketone,2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol, propylacetate, 1-1,diethoxypropane, 1,1-dimethoxymethane,2,2-dimethyoxypropane, isootcane, isopropyl ether, methylisopropylketone, methyltetrahydrofuran, petroleum ether, trichloroacetic acid,and trifluoroacetic acid. Additionally, the drying process may beconducted at a temperature of about 28° C. or less, alternatively at atemperature of about 26° C. or less, alternatively at a temperature ofabout 24° C. or less, alternatively at a temperature of about 22° C. orless, alternatively at a temperature of about 20° C. or less,alternatively at a temperature of about 15° C. or less, alternatively ata temperature of about 110° C. or less. The vacuum pressure of thedrying procedure may be about 50 mbar or less, alternatively about 45mbar or less, alternatively about 40 mbar or less, alternatively about35 mbar or less, alternatively about 30 mbar or less, alternativelyabout 25 mbar or less, alternatively about 20 mbar or less,alternatively about 15 mbar or less, alternatively about 10 mbar orless, alternatively about 5 mbar or less. Additionally, the water levelmay be less than about 25% (w/w), alternatively less than about 20%(w/w), alternatively less than about 15% (w/w).

In addition to the low solvent content, the drying process may alsoreduce the amount of MAG present in the dalbavancin composition. Theamount of MAG present may be below about 5% HPLC distribution,alternatively below about 4.5% HPLC distribution, alternatively belowabout 4.0% HPLC distribution, alternatively below about 3.5% HPLCdistribution, alternatively below about 3.0% HPLC distribution,alternatively below about 2.5% HPLC distribution, alternatively belowabout 2.0% HPLC distribution, alternatively below about 1.5% HPLCdistribution, alternatively below about 1.0% HPLC distribution,alternatively below about 0.8% HPLC distribution, alternatively belowabout 0.6% HPLC distribution, alternatively below about 0.5% HPLCdistribution, alternatively below about 0.4% HPLC distribution,alternatively below about 0.3% HPLC distribution, alternatively belowabout 0.2% HPLC distribution, alternatively below about 0.1% HPLCdistribution.

The invention also includes a pharmaceutical composition comprisingdalbavancin factors B₂ and isoB₀. The content of each of B₂ and isoB₀can independently not exceed about 3.0 percent HPLC distribution,alternatively can independently not exceed about 2.5 percent HPLCdistribution, alternatively can independently not exceed about 2.0percent HPLC distribution, alternatively can independently not exceedabout 1.5 percent HPLC distribution, alternatively can independently notexceed about 1.0 percent HPLC distribution, alternatively canindependently not exceed about 0.5 percent HPLC distribution, oralternatively can independently not exceed about 0.1 percent HPLCdistribution.

Additionally, the invention also includes a pharmaceutical compositioncomprising dalbavancin factors B₂, isoB₀, and MAG. The content of eachof B₂, isoB₀, and MAG can independently not exceed about 3.0 percentHPLC distribution, alternatively can independently not exceed about 2.5percent HPLC distribution, alternatively can independently not exceedabout 2.0 percent HPLC distribution, alternatively can independently notexceed about 1.5 percent HPLC distribution, alternatively canindependently not exceed about 1.0 percent HPLC distribution,alternatively can independently not exceed about 0.5 percent HPLCdistribution, or alternatively can independently not exceed about 0.1percent HPLC distribution.

The invention also includes methods for treating a bacterial infectioncomprising providing at least one of the pharmaceutical compositionsdescribed above to patient in need thereof and administering atherapeutically effective dose of sterile, stable, particle-free, cleardalbavancin to the patient. The method may further include administeringa single subsequent therapeutically effective dose. The singlesubsequent therapeutically effective dose may be administeredapproximately five to ten days, or about a week, after the initial dose.The single subsequent therapeutically effective dose may also beadministered approximately five to ten days, or about a week, after theinitial dose, without any intervening dose of dalbavancin. In anotherembodiment, the method may include administering multiple subsequentdoses. The multiple subsequent doses may be administered atapproximately five to ten day intervals, or one week intervals. Themultiple subsequent doses may also be administered at approximately fiveto ten day intervals, or one week intervals, without any interveningdoses of dalbavancin. The method may also include the further step ofmonitoring the infection after administering the first dose and,optionally, adjusting the subsequent dose(s) accordingly.

The invention also includes methods for treating a bacterial infectionin a patient with renal impairment comprising administering atherapeutically effective dose of sterile, stable, particle-free, cleardalbavancin to the patient. The impairment can range from mild tosevere. In one embodiment, the therapeutically effective dose achieves apeak concentration in the patient (C_(max)) of at least 100 mg/L. Inanother embodiment, the therapeutically effective dose achieves apatient exposure (area under the curve) of at least 13,000 mg·h/L. Themethod may include administering a single dose of dalbavancin of about300-1200 mg, alternatively about 400 mg, alternatively about 500 mg,alternatively about 600 mg, alternatively about 700 mg, alternativelyabout 800 mg, alternatively about 900 mg, alternatively about 1000 mg,alternatively about 1100 mg, alternatively about 1200 mg.

The method for treating a bacterial infection in a patient with renalimpairment may also include administering multiple doses of dalbavancin.In one embodiment, two doses may be administered about five to about tendays apart, such as about one week apart, or alternatively about ten toabout eighteen days apart, such as a bout two weeks (or 14 days apart).Alternatively, dose frequency may be, for example, twice weekly doses,thrice weekly doses, or multiple weekly doses. Alternatively, the dosinginterval may be, for example, any of about 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more daysapart. The number of doses given, can be, for example, one, two, three,four, five, six, or more doses, each dose after the initial dose beinggiven after the selected dosage interval.

In one embodiment, the first dose may be about 750 mg and the second (orsubsequent) dose may be about 250 mg. In another embodiment, the firstdose may be about 750 mg and the second (or subsequent) dose may beabout 150 mg. In another embodiment, the first dose may be about 750 mgand the second (or subsequent) dose may be about 125 mg. In anotherembodiment, the first dose may be about 1000 mg and the second (orsubsequent) dose may be about 500 mg.

In another embodiment, the first dose may be about 200 mg to about 1500mg, alternatively about 200 mg to about 1300 mg, alternatively about 100mg to about 1500 mg, alternatively about 200 mg to about 1400 mg,alternatively about 300 mg to about 1300 mg, alternatively about 400 mgto about 1200 mg, alternatively about 500 mg to about 1100 mg,alternatively about 600 mg to about 1000 mg, alternatively about 1000mg, alternatively about 950 mg, alternatively about 900 mg,alternatively about 850 mg, alternatively about 800 mg, alternativelyabout 750 mg, alternatively about 700 mg, alternatively about 650 mg,alternatively about 600 mg, alternatively about 550 mg, or alternativelyabout 500 mg. The second or subsequent dose may be about 200 mg to about1500 mg, alternatively about 200 mg to about 1300 mg, alternativelyabout 100 mg to about 1500 mg, alternatively about 200 mg to about 1400mg, alternatively about 300 mg to about 1300 mg, alternatively about 400mg to about 1200 mg, alternatively about 500 mg to about 1100 mg, oralternatively about 600 mg to about 1000 mg, alternatively about 600 mg,alternatively about 550 mg, alternatively about 500 mg, alternativelyabout 450 mg, alternatively about 400 mg, alternatively about 350 mg,alternatively about 300 mg, alternatively about 250 mg, alternativelyabout 200 mg, alternatively about 150 mg, or alternatively about 100 mg.It is to be understood that any of the above first doses may be combinedwith any of the above second or subsequent doses in a dosing regimen.

In a multiple dosing regimen for renal or normal patients, the ratiobetween the first and second (or subsequent) doses may be defined by afactor. The amount of the first dose may be greater than about 1 timesthe amount of the second dose. Alternatively, the first dose may begreater than about 1.5 times, greater than about 2 times, greater thanabout 2.5 times, greater than about 3 times, greater than about 3.5times, greater than about 4 times, greater than about 4.5 times, greaterthan about 5 times, greater than about 5.5 times, or greater than about6 times the amount of the second (or subsequent) doses.

Similarly, the amount of the second (or subsequent) dose may be about 6times less than the amount of the first dose. Alternatively, the amountof the second (or subsequent) dose may be about 5.5 times, alternativelyless than about 5.0 times, alternatively less than about 4.5 times,alternatively less than about 4.0 times, alternatively less than about3.5 times, alternatively less than about 3.0 times, alternatively lessthan about 2.5 times, alternatively less than about 2.0 times,alternatively less than about 1.5 times, alternatively less than about1.0 times the amount of the first dose.

The method for treating a bacterial infection in a patient with renalimpairment may include administering a single dose of dalbavancin. Thepatient may have mild, moderate, severe, or end-stage renal impairment.The amount of this single dose may be about 1200 mg, alternatively about1150 mg, alternatively about 1100 mg, alternatively about 1050 mg,alternatively about 1000 mg, alternatively about 950 mg, alternativelyabout 900 mg, alternatively about 850 mg, alternatively about 800 mg,alternatively about 750 mg, alternatively about 700 mg, alternativelyabout 650 mg, alternatively about 600 mg, alternatively about 550 mg,alternatively about 500 mg. The amount of the single dose may also bebetween about 250 mg to about 1300 mg, alternatively between about 300mg to about 1200 mg, alternatively between about 350 mg to about 1100mg, alternatively between about 400 mg to about 1000 mg, alternativelybetween about 450 mg to about 1000 mg, alternatively between about 500mg to about 1000 mg, alternatively between about 550 mg to about 950 mg,alternatively between about 600 mg to about 900 mg, alternativelybetween about 650 mg to about 850 mg.

The invention also includes methods of making the pharmaceuticalcompositions described above comprising providing dalbavancin and addinga stabilizer. In one embodiment, the stabilizer is a carbohydrate or asugar. In another embodiment, the stabilizer is mannitol, lactose, or acombination thereof.

In yet another embodiment, the method further includes the step ofadjusting the pH of the composition, if necessary. In one embodiment,the pH is adjusted to about 1-7, more preferably about 2-6, morepreferably about 3-5. The pH may be adjusted with a pH modifier. pHmodifiers include inorganic bases of alkali and alkaline earth metalssuch as NaOH, Ca(OH)₂, KOH, and Mg(OH)₂. Inorganic oxides, carbonates,bicarbonates of alkali and alkaline earth metals may also be used as pHmodifiers. Alkali and alkaline salts of inorganic weak acids andamphoteric acids such as phosphoric acid, boric acid, sulfuric acid, andall other sulfur-containing acids may also be used as pH modifiers.Amino acids such as lysine, meglumaine, arginine, n-methyl glucosamine,may also be used to modify the pH. Organic bases, including all amines,phenols, weak carboxylic acids, dicarboxylic acids, hydroxy carboxylicacids, and their salts, may also be utilized to modify the pH of thecomposition.

In another aspect, methods are provided for treating a bacterialinfection in an individual in need thereof, including administering atleast one unit dose of dalbavancin in an amount sufficient to provide atherapeutically effective plasma level of dalbavancin in the individualfor at least five days, and a pharmaceutically acceptable carrier. Atherapeutically effective plasma level of dalbavancin is generally atleast about 4 mg of dalbavancin per liter of plasma. In one embodiment,the dosage amount of dalbavancin administered is an amount that isclinically effective and also has reduced adverse side effects incomparison to the standard of care with drugs such as teicoplanin andvancomycin.

Dalbavancin may be administered as a single dose or as multiple doses.In some embodiments, a single dose of about 100 mg to about 4000 mg, forexample 3000 mg, of dalbavancin is administered. In various embodiments,a single dalbavancin dose may include at least about any of 0.1, 0.25,0.5, 1, 1.5, 2, 2.5, or 3 grams.

In other embodiments, two doses are administered about five to about tendays apart, such as about one week apart. The first dose may be about500 to about 5000 mg of dalbavancin and the second dose may be about 250mg to about 2500 mg of dalbavancin. Often, the first dose includes about1.5 to about 3 times, often at least about twice as much of the amountof dalbavancin contained in the second dose. For example, the first dosemay be about 1000 mg and the second dose may be about 500 mg ofdalbavancin. In another embodiment, the first dose may be about 1200 mgand the second dose may be about 600 mg. In methods in which two dosesare administered, the plasma trough level of dalbavancin in anindividual prior to administration of the second dose is generally atleast about 4 mg, often at least about 10 mg, often at least about 20mg, more often at least about 30 mg dalbavancin per liter of plasma, andstill more often at least about 40 mg dalbavancin per liter of plasma.

Often, methods of the invention include parenteral administration, forexample intravenous administration. In some embodiments, administrationis intravenous with the rate of administration controlled such thatadministration occurs over at least about 30 minutes or longer.

Methods of the invention may be used to treat a Gram-positive bacterialinfection, such as, for example, a Staphylococcus aureus orStreptococcus pyogenes skin and soft tissue infection. In someembodiments, the infection is penicillin-resistant and/or multi-drugresistant.

In another aspect, a method for preventing a bacterial infection isprovided which includes administering at least one unit dose ofdalbavancin in an amount sufficient to provide a prophylacticallyeffective plasma level of dalbavancin in the individual for at leastabout one day, three days, five days, one week, or ten days or longer,and a pharmaceutically acceptable carrier. The dosage of dalbavancin maybe, for example, about 100 mg to about 1000 mg. In some embodiments,dalbavancin is administered prior, during, or subsequent to a medicalprocedure or a stay in the hospital.

Therapeutic or prophylactic methods of the invention may includeadministration of at least one antibiotic that is not dalbavancin,preferably an antibiotic that is effective against a Gram-negativebacterium and/or an antibiotic that is effective against Gram-positivestrains that dalbavancin is not effective against, such as VanA strains.

In another aspect, kits are provided that include at least one unit doseof dalbavancin in an amount sufficient to provide a therapeuticallyeffective plasma level for at least about five days or aprophylactically effective plasma level of dalbavancin for at leastabout one day in an individual, and instructions for use in a method oftreatment or prophylaxis of a bacterial infection. A kit may contain twounit dosages, with a first dosage including 1.5 to 3 times, often atleast about twice as much of the amount of dalbavancin included in asecond dosage. Kits may also include an antibiotic that is notdalbavancin, preferably effective against a Gram-negative bacterium.

In one embodiment, kits are provided that include a first containercontaining a dry powder (e.g., lyophilized) dalbavancin composition anda second container containing a predetermined amount of aphysiologically acceptable aqueous solution for admixing with thedalbavancin composition. Such solutions are preferably sterile aqueoussolutions. In one embodiment, kits include a delivery means foradministering the dalbavancin composition to an individual, for examplea syringe or intravenous administration means.

The present invention also provides N¹⁵,N¹⁵-dialkyl antibiotic compoundswith utility against infectious microbes. The compounds of the inventionare useful for treating and/or preventing microbial infections, such asSSTIs and other bacterial infections.

The present invention is based, in part, on the discovery ofN¹⁵,N¹⁵-dialkyl antibiotic compounds having antimicrobial activity atleast comparable to, or even greater than, previously knownN¹⁵-monoalkyl antibiotic compounds such as dalbavancin compounds. Inaddition, as described in the examples below, certain N¹⁵,N¹⁵-dialkylantibiotic compounds of the invention show activity against a broaderspectrum of microbes when compared to the previously known N¹⁵-monoalkylantibiotic compounds such as N¹⁵-monomethyl dalbavancin compounds.

In one aspect, the present invention provides N¹⁵,N¹⁵-dialkyl antibioticcompounds according to formula (I):

The N¹⁵,N¹⁵-dialkyl antibiotic compounds of the invention (having atomsnumbered according to FIG. 26) comprise two alkyl substituents on theamino terminal nitrogen. In other words, in formula (I) R¹ and R^(1′)are alkyl. In most preferred embodiments of the invention, R¹ and R^(1′)are methyl. The other substituents of formula (I) are described indetail below. Preferred substituents include those found on antibiotic A40926 compounds and/or dalbavancin compounds known to those of skill inthe art. For instance, in preferred embodiments G and M are glycosylmoieties such as the aminoglucuronyl and mannopyranosyl moietiesdescribed in the sections below. In certain embodiments, theaminoglucuronyl moiety is acylated, for example, with a fatty acid, andin certain embodiments, the mannopyranosyl moiety is acetylated.

In certain embodiments, X is OH while in further embodiments X isaminoalkylamino. The aminoalkylamino group can be any aminoalkylaminogroup known to those of skill in the art, including those described inU.S. Pat. No. 5,750,509. In preferred embodiments, X isN,N-dimethylaminopropylamino. As described in detail in the examplesbelow, compounds according to formula (I) wherein X is aminoalkylaminoshow antibiotic activity comparable to or greater than correspondingdalbavancin compounds, such as those described in U.S. Pat. No.5,750,509. Although compounds wherein X is aminoalkylamino arepreferred, compounds wherein X is OH are useful, for example, for thepreparation of compounds of the invention wherein X is aminoalkylamino,and they can also be useful themselves for treating and/or preventingmicrobial infections.

In another aspect, the present invention provides compositionscomprising an N¹⁵,N¹⁵-dialkyl antibiotic compound of the invention and asecond compound. The second compound can be any compound known to thoseof skill in the art. In particular embodiments, the second compound isan antibiotic compound, for instance an antibiotic A 40926 compound or adalbavancin compound. In further embodiments, the second compound isalso an N¹⁵,N¹⁵-dialkyl antibiotic compound of the invention. Exemplarycompositions of the invention include mixtures of antibiotic A 40926compounds and/or dalbavancin compounds that further comprise a compoundaccording to formula (I). In particular embodiments, the compositionsare enriched in the N¹⁵,N¹⁵-dialkyl antibiotic compound of theinvention. The enrichment can be relative to antibiotic A 40926compounds and/or dalbavancin compounds of the composition, or relativeto other compounds of the composition, or both. In certain embodiments,the present invention provides compositions comprising a purifiedN¹⁵,N¹⁵-dialkyl antibiotic compound of the invention, an isolatedN¹⁵,N¹⁵-dialkyl antibiotic compound of the invention or a purified andisolated N¹⁵,N¹⁵-dialkyl antibiotic compound of the invention.

In a further aspect, the present invention provides pharmaceuticalcompositions comprising a N¹⁵,N¹⁵-dialkyl antibiotic compound of theinvention. Preferred compositions comprise compounds according toformula (I) wherein X is aminoalkylamino The compositions can furthercomprise other active ingredients, including antibiotic A 40926compounds and/or dalbavancin compounds known to those of skill in theart. In certain embodiments, the pharmaceutical compositions furthercomprise a pharmaceutically acceptable carrier, excipient or diluent. Inparticular embodiments, the present invention provides pharmaceuticalunit dosages of a compound of the invention for use, for example, in thetreatment and/or prevention of microbial infections.

In a further aspect, the present invention provides a dosage formcomprising a sterile, stable, particle-free dalbavancin powder suitablefor reconstitution with a pharmaceutically acceptable vehicle comprisingdalbavancin factors B₀ and B₂. The names B₂, C₂, and N¹⁵,N¹⁵-dimethyldalbavancin B₀ have been used interchangeably. In one embodiment, thecontent of factor B₀ is not less than about 75 percent HPLCdistribution, alternatively not less than about 80 percent HPLCdistribution, alternatively not less than about 85 percent HPLCdistribution, alternatively not less than about 90 percent HPLCdistribution, alternatively not less than about 95 percent HPLCdistribution. The content of B₂ is not less than about 4.0 percent HPLCdistribution, alternatively not less than about 5.0 percent HPLCdistribution, alternatively not less than about 6.0 percent HPLCdistribution, alternatively not less than about 7.0 percent HPLCdistribution, alternatively not less than about 8.0 percent HPLCdistribution, alternatively not less than about 9.0 percent HPLCdistribution, alternatively not less than about 10.0 percent HPLCdistribution, alternatively not less than about 15.0 percent HPLCdistribution, alternatively not less than about 20.0 percent HPLCdistribution, alternatively not less than about 25.0 percent HPLCdistribution, alternatively not less than about 30.0 percent HPLCdistribution, alternatively not less than about 40.0 percent HPLCdistribution. The percent HPLC distribution of particular components canbe calculated by comparing the area of each single component to thetotal chromatography area. The dosage form may further include at leastone additional factor, such as dalbavancin factor A₀, A₁, B₁, C₀, or C₁.

In a further aspect, the present invention provides for a pharmaceuticalcomposition comprising dalbavancin factors B₀ and B₂. In one embodiment,the content of factor B₀ is not less than about 75 percent HPLCdistribution, alternatively not less than about 80 percent HPLCdistribution, alternatively not less than about 85 percent HPLCdistribution, alternatively not less than about 90 percent HPLCdistribution, alternatively not less than about 95 percent HPLCdistribution. The content of B₂ is not less than about 3.0 percent HPLCdistribution, alternatively not less than about 4.0 percent HPLCdistribution, alternatively not less than about 5.0 percent HPLCdistribution, alternatively not less than about 6.0 percent HPLCdistribution, alternatively not less than about 7.0 percent HPLCdistribution, alternatively not less than about 8.0 percent HPLCdistribution, alternatively not less than about 9.0 percent HPLCdistribution, alternatively not less than about 10.0 percent HPLCdistribution, alternatively not less than about 15.0 percent HPLCdistribution, alternatively not less than about 20.0 percent HPLCdistribution, alternatively not less than about 25.0 percent HPLCdistribution, alternatively not less than about 30.0 percent HPLCdistribution, alternatively not less than about 40.0 percent HPLCdistribution. As defined above, the percent HPLC distribution ofparticular components can be calculated by comparing the area of eachsingle component to the total chromatography area. The pharmaceuticalcomposition may further include at least one additional factor, such asdalbavancin factor A₀, A₁, B₁, C₀, or C₁.

In another aspect, the present invention also provides for thede-enrichment of the B₂ component. In one embodiment, the content offactor B₂ is not more than about 5.0 percent HPLC distribution,alternatively not more than about 4.5 percent HPLC distribution,alternatively not more than about 4.0 percent HPLC distribution,alternatively not more than about 3.5 percent HPLC distribution,alternatively not more than about 3.0 percent HPLC distribution,alternatively not more than about 2.5 percent HPLC distribution,alternatively not more than about 2.0 percent HPLC distribution,alternatively not more than about 1.5 percent HPLC distribution,alternatively not more than about 1.0 percent HPLC distribution,alternatively not more than about 0.5 percent HPLC distribution,alternatively not more than about 0.1 percent HPLC distribution.

In another aspect, the present invention provides methods of treatingand/or preventing microbial infections in a subject in need thereof. Themethods can comprise the administration of an effective amount of aN¹⁵,N¹⁵-dialkyl antibiotic compound or composition of the invention tothe subject. The invention encompasses the prevention or treatment ofgram-positive or antibiotic-resistant bacterial infections, such as aBacillus, Corynebacteria, Listeria, Enterococcus, Staphylococcus,Streptococcus, Neisseria, or Clostridium genus infection, in particularStaphylococcus aureus, Staphylococcus epidermidis, Staphylococcushemolyticus, Streptococcus pyogenes, Streptococcus pneumoniae, Groups Aand C Streptococcus, Enterococcus faecalis, Bacillus subtilis, Neisseriagonorrhoeae, or Clostridium difficile. Other infections that can beprevented or treated using the N¹⁵,N¹⁵-dialkyl antibiotic compounds,compositions, and methods of the invention include gram negativebacterial infections, such as a Bartonella, Brucella, Campylobacter,Enterobacter, Escherichia (as well as other Proteobacteria),Francisella, Helicobacter, Hemophilus, Klebsiella, Legionella,Leptospira, Morganella, Moraxella, Proteus, Providencia, Pseudomonas,Salmonella, Serratia, Shigella, Stenotrophomonas, Vibrio, and Yersiniagenus infection, in particular Escherichia coli, Proteus vulgaris,Pseudomonas aeruginosas, and yeast, such as Candida albicans,infections.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts the amount of dalbavancin component B₀ versus time invarious pharmaceutical compositions, with and without mannitol, at 25°C.

FIG. 1B depicts the amount of MAG versus time in various pharmaceuticalcompositions, with and without mannitol, at 25° C.

FIG. 2A depicts the amount of dalbavancin component B₀ versus time invarious pharmaceutical compositions, with and without mannitol, at 40°C.

FIG. 2B depicts the amount of MAG versus time in various pharmaceuticalcompositions, with and without mannitol, at 40° C.

FIG. 3A depicts the amount of dalbavancin component B₀ versus time invarious pharmaceutical compositions containing mannitol and/or lactoseat 25° C.

FIG. 3B depicts the amount of MAG versus time in various pharmaceuticalcompositions containing mannitol and/or lactose at 25° C.

FIG. 4A depicts the amount of dalbavancin component B₀ versus time invarious pharmaceutical compositions containing mannitol and/or lactoseat 40° C.

FIG. 4B depicts the amount of MAG versus time in various pharmaceuticalcompositions containing mannitol and/or lactose at 40° C.

FIG. 5 depicts dalbavancin plasma concentration versus time following asingle 1000 mg intravenous infusion of dalbavancin.

FIG. 6 depicts isothermal titration calorimetry data for dalbavancinbinding to human serum albumin (top) and a graphical representation ofthe data fitted to a curve determined from a 2:1 binding model ofdalbavancin:protein (bottom).

FIG. 7 depicts an electrospray ionization mass spectrum of dalbavancin.

FIG. 8 is a graph of dalbavancin concentration vs. population ratio ofdalbavancin multimer to monomer and depicts an increase in populationratio of dalbavancin multimer to monomer with increasing dalbavancinconcentration.

FIG. 9 is a graph of pH vs. population ratio of dalbavancin multimer tomonomer and depicts an increase in population ratio of dalbavancinmultimer to monomer with increasing pH.

FIG. 10 depicts an electrospray ionization mass spectrum of dalbavancinin an ammonium formate 5 mM pH 5 solution.

FIG. 11 depicts an electrospray ionization mass spectrum of dalbavancinin an ammonium formate 50 mM pH 5 solution.

FIG. 12 depicts an electrospray ionization mass spectrum of dalbavancinin an ammonium formate 100 mM pH 5 solution.

FIG. 13 depicts an electrospray ionization mass spectrum of teicoplanin(50 μg/mL) in water.

FIG. 14 depicts an electrospray ionization mass spectrum of teicoplanin(100 μg/mL) in water.

FIG. 15 depicts the effect of HSA on the apparent dissociation constantfor dalbavancin/tri-peptide binding at 26° C. (pH 7.4).

FIG. 16 depicts a comparison of isothermal calorimetry (ITC) data forbinding of tri-peptide to vancomycin and dalbavancin under identicalconditions, using the same tri-peptide solution.

FIGS. 17A and 17B depict the possible interaction of dalbavancinmonomers and multimers (including dimers) with tri-peptide ligand andHSA. FIG. 17A depicts dalbavancin in monomer-dimer equilibrium insolution, binding as monomer to two separate sites on HSA. FIG. 17Bdepicts ligand binding to dalbavancin dimer in solution and more weaklyto dalbavancin monomers attached to HAS

FIG. 18 provides mean dalbavancin plasma concentration-time profiles.

FIG. 19 provides mean dalbavancin plasma concentration-time profiles.

FIG. 20 provides additional mean dalbavancin plasma concentration-timeprofiles.

FIG. 21 provides a graph of dalbavancin exposure through the relativetreatment period vs. creatinine clearance.

FIG. 22 provides a comparison of exposure during the relative treatmentperiod vs. overall exposure.

FIG. 23 provides dalbavancin concentration-time profiles for subjectswith normal renal function and subjects with severe renal impairment.

FIG. 24 provides dalbavancin plasma concentration time profiles.

FIG. 25 provides a comparison of dalbavancin exposure through thetreatment period.

FIG. 26 provides the structure of antibiotic A 40926 compounds anddalbavancin compounds including the numbering of selected atoms;

FIG. 27 provides the structure of dalbavancin B₀;

FIG. 28 provides the structure of an exemplary N¹⁵,N¹⁵-dimethylantibiotic compound of the invention;

FIGS. 29-31 provide schemes illustrating examples of the invention thatverify the structure of an N¹⁵,N¹⁵-dimethyl antibiotic compound of theinvention;

FIGS. 32-33 provide ESI and HPLC chromatograms verifying the structureof the N¹⁵,N¹⁵-dimethyl antibiotic compound;

FIG. 34 provides the structure of dalbavancin B₂;

FIG. 35 provides an infrared spectrum of dalbavancin B₂(N¹⁵,N¹⁵-dimethyl dalbavancin B₀);

FIG. 36 provides an ESI-MS spectrum of dalbavancin B₂ (N¹⁵,N¹⁵-dimethyldalbavancin B₀); and

FIG. 37 illustrates an HPLC trace of a composition of the invention.

FIG. 38 provides a mass spectrum of isoB₀.

FIG. 39 illustrates an HPLC trace of isoB₀.

FIGS. 40A-C provides mass spectra of isoB₀.

FIG. 41 provides a ¹H-NMR spectrum of isoB₀.

FIG. 42 provides a ¹³C-NMR spectrum of isoB₀.

FIG. 43 provides the identification of the location of the protons fordalbavancin.

FIG. 44 provides the plasma pharmacokinetics of dalbavancin inthigh-infected mice.

FIG. 45 provides the effect of single doses of dalbavancin on the invivo killing of S. pneumoniae over time.

FIG. 46 provides the effect of single doses of dalbavancin on the invivo killing of S. aureus over time.

FIG. 47 provides the relationship between dalbavancin dosing intervaland efficacy against S. pneumoniae.

FIG. 48 provides the relationship between dalbavancin dosing intervaland efficacy against S. aureus.

FIG. 49 provides the relationship between dalbavancin PK/PD parametersand efficacy against S. pneumoniae.

FIG. 50 provides the relationship between dalbavancin PK/PD parametersand efficacy against S. aureus.

FIG. 51 provides the dose-response curves for dalbavancin aginst variousstrains.

FIG. 52 provides the dose-response curves for dalbavancin aginst S.pneumoniae and S. aureus.

FIG. 53 provides the dose-response curves for dalbavancin in both normaland neutropenic mice infected with S. pneumoniae.

FIG. 54 provides the dose-response curves for dalbavancin in both thelung and thigh infection models.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

When describing compounds of the invention, pharmaceutical compositionscontaining such compounds and methods of using such compounds andcompositions, the following terms have the following meanings unlessotherwise indicated.

“Antibiotic A 40926 compound” refers to a glycopeptide antibiotic knownto those of skill in the art. Exemplary antibiotic A 40926 compounds aredescribed in U.S. Pat. Nos. 4,935,238, 4,868,171 and 4,782,042, thecontents of which are hereby incorporated by reference in theirentireties.

“Dalbavancin compound” refers to a glycopeptide antibiotic described inU.S. Pat. No. 5,750,509 and U.S. Patent Application Publication No.2004/0142883, the contents of which are hereby incorporated by referencein their entireties. The systematic name of an exemplary dalbavancincompound known to those of skill in the art is ristomycin A aglycone,5,31-dichloro-38-de(methoxycarbonyl)-7-demethyl-19-deoxy-56-O-(2-deoxy-2-((10-methyl-1-oxoundecyl)amino)-beta-D-glucopyranuronosyl)-38-(((3-(dimethylamino)propyl)amino)-carbonyl)-42-O-alpha-D-mannopyranosyl-N¹⁵-methyl-.Examples include those depicted in FIGS. 26 and 27. Dalbavancincompounds include those compounds with optional sugar moieties at the 56and 42 positions and optional acyl groups, such as fatty acids, on thesugars. Dalbavancin compounds can be derived from the natural antibioticA-40926 known to those of skill in the art. Exemplary dalbavancincompounds include those described U.S. Patent Application PublicationNo. 2004/0142883 such as dalbavancin A₀, A₁, B₀, B₁, C₀ and C₁.

“N¹⁵,N¹⁵-dialkyl antibiotic compound,” described in detail in thesections below, refers to a compound of the invention, i.e. anantibiotic compound having two alkyl groups on its N¹⁵ nitrogen. AN¹⁵,N¹⁵-dialkyl antibiotic compound of the invention can be derived froman antibiotic A 40926 compound or from a dalbavancin compound such asdalbavancin A₀, A₁, B₀, B₁, C₀ and C₁.

“N¹⁵,N¹⁵-dimethyl antibiotic compound,” described in detail in thesections below, refers to a compound of the invention, i.e. anantibiotic compound, having two methyl groups on its N¹⁵ nitrogen.

“Acyl” refers to a radical —C(O)R, where R is alkyl.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groupspreferably having from 1 to about 11 carbon atoms, more preferably from1 to 8 carbon atoms, and still more preferably from 1 to 6 carbon atoms.The hydrocarbon chain may be either linear or branched. This term isexemplified by groups such as methyl, ethyl, n-propyl, isopropyl,n-butyl, iso-butyl, tert-butyl, n-hexyl, n-octyl, tert-octyl and thelike. The term “lower alkyl” refers to alkyl groups having from 1 to 6carbon atoms.

“Alkylene” refers to divalent saturated aliphatic hydrocarbyl groupspreferably having from 1 to 11 carbon atoms and more preferably 1 to 6carbon atoms which can be linear or branched. This term is exemplifiedby groups such as methylene (—CH₂—), ethylene (—CH₂CH₂—), the propyleneisomers (e.g., —CH₂CH₂CH₂— and —CH(CH₃)CH₂—) and the like.

“Amino” refers to the radical —NH₂.

“Alkylamino” refers to the radical —NH-alkyl or —N(alkyl)₂.

“Aminoalkylamino” refers to a radical of the form —NR-(alkyl)-NR′R″wherein each R, R′ and R″ independently represent hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, cycloalkyl, substitutedcycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroarylor substituted heteroaryl group. Preferred R, R′ and R″ groups includehydrogen and alkyl. Exemplary aminoalkylamino groups are described inU.S. Pat. No. 5,750,509, the contents of which are hereby incorporatedby reference in their entirety.

“Carboxy” refers to the radical —C(O)OH.

“Dialkylamino” means a radical —NRR′ where R and R′ each independentlyrepresent an alkyl, substituted alkyl, aryl, substituted aryl,cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substitutedcycloheteroalkyl, heteroaryl or substituted heteroaryl group as definedherein.

“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo. Preferredhalo groups are either fluoro or chloro.

“Percent HPLC distribution” of a particular component is cacluated bycomparing the area of the peak corresponding to the particular componentto the total chromatographic area.

“Pharmaceutically acceptable” means approved (by a regulatory agency forinvestigational or commercial use) of the federal or a state governmentor listed in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals, and more particularly in humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound of theinvention that is pharmaceutically acceptable and that possesses thedesired pharmacological activity of the parent compound. Such saltsinclude: (1) acid addition salts formed with organic or inorganic acidssuch as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, acetic,trifluoroacetic, trichloroacetic, propionic, hexanoic,cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic,succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric,benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic,phthalic, lauric, methanesulfonic, ethanesulfonic,1,2-ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic,4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic,camphoric, camphorsulfonic,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic, glucoheptonic,3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric,gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic,muconic acid and the like acids; or (2) salts formed when an acidicproton present in the parent compound either (a) is replaced by a metalion, e.g., an alkali metal ion, an alkaline earth ion or an aluminumion, or alkali metal or alkaline earth metal hydroxides, such as sodium,potassium, calcium, magnesium, and barium hydroxide, ammonia or (b)coordinates with an organic base, such as aliphatic, alicyclic, oraromatic organic amines, such as methylamine, dimethylaminem,diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine,N-methylglucamine and the like (see, e.g., U.S. Pat. No. 5,606,036).

Salts further include, by way of example only, sodium, potassium,calcium, magnesium, ammonium, tetraalkylammonium and the like, and whenthe compound contains a basic functionality, salts of non-toxic organicor inorganic acids, such as hydrochloride, hydrobromide, tartrate,mesylate, acetate, maleate, oxalate and the like. The term“pharmaceutically acceptable cation” refers to a non-toxic,pharmaceutically acceptable cationic counterion of an acidic functionalgroup. Such cations are exemplified by sodium, potassium, calcium,magnesium, ammonium and tetraalkylammonium cations and the like.

“Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant,excipient or carrier with which a compound of the invention isadministered.

“Solvate” refers to a compound of the present invention or a saltthereof, that further includes a stoichiometric or non-stoichiometricamount of solvent bound by non-covalent intermolecular forces. Where thesolvent is water, the solvate is a hydrate.

“Preventing” or “prevention” refers to a reduction in the risk ofacquiring a disease or disorder (i.e., causing at least one of theclinical symptoms of the disease not to develop in a subject that may beexposed to or predisposed to the disease but does not yet experience ordisplay symptoms of the disease). Preferably, prevention refers to theuse of a compound or composition in a subject not yet affected by thedisease or disorder or not yet exhibiting a symptom of the disease ordisorder, for instance a subject not yet infected or not yet exhibitingthe symptoms of infection.

“Subject” includes humans. The terms “human,” “patient” and “subject”are used interchangeably herein.

“Therapeutically effective amount” means an amount of a compound orcomposition that, when administered to a subject for treating a disease,is sufficient to effect such treatment for the disease. A“therapeutically effective amount” can vary depending on, inter alia,the compound, the disease and its severity, and the age, weight, etc.,of the subject to be treated.

“Treating” or “treatment” of any disease or disorder refers, in oneembodiment, to ameliorating the disease or disorder (i.e., arresting orreducing the development of the disease or at least one of the clinicalsymptoms thereof) that exists in a subject. In another embodiment,“treating” or “treatment” refers to ameliorating at least one physicalparameter, which may be indiscernible by the subject. In yet anotherembodiment, “treating” or “treatment” refers to modulating the diseaseor disorder, either physically (e.g., stabilization of a discerniblesymptom) or physiologically (e.g., stabilization of a physicalparameter) or both. In yet another embodiment, “treating” or “treatment”refers to delaying the onset of the disease or disorder.

It is to be understood that compounds having the same molecular formulabut differing in the nature or sequence of bonding of their atoms or inthe arrangement of their atoms in space are termed “isomers”. Isomersthat differ in the arrangement of their atoms in space are termed“stereoisomers”.

Stereoisomers that are not mirror images of one another are termed“diastereomers” and those that are non-superimposable mirror images ofeach other are termed “enantiomers”. When a compound has an asymmetriccenter, for example, when it is bonded to four different groups, a pairof enantiomers is possible. An enantiomer can be characterized by theabsolute configuration of its asymmetric center and is designated (R) or(S) according to the rules of Cahn and Prelog, or can be characterizedby the manner in which the molecule rotates the plane of polarized lightand is designated dextrorotatory or levorotatory (i.e., as (+)- or(−)-isomers, respectively). A chiral compound can exist as eitherindividual enantiomer or as a mixture thereof. A mixture containingequal proportions of enantiomers is called a “racemic mixture”.

In certain embodiments, the compounds of this invention may possess oneor more asymmetric centers; such compounds can therefore be produced asthe individual (R)- or (S)-enantiomer or as a mixture thereof. Unlessindicated otherwise, for example by designation of stereochemistry atany position of a formula, the description or naming of a particularcompound in the specification and claims is intended to include bothindividual enantiomers and mixtures, racemic or otherwise, thereof.Methods for determination of stereochemistry and separation ofstereoisomers are well-known in the art. In particular embodiments, thepresent invention provides the stereoisomers of the compounds depictedherein upon treatment with base.

The present invention provides novel pharmaceutical compositions ofdalbavancin, methods of making the pharmaceutical compositions, andmethods of treatment of bacterial infections using these novelcompositions. In particular, the invention provides stable dalbavancincompositions having bactericidal activity, which may be refrigerated orstored at room temperature for a prolonged period of time, morepreferably at least one year at room temperature, more preferably atleast two years at room temperature, without significant degradation ofthe active dalbavancin component.

The present invention also provides improved dosage regimes and novelcompositions of dalbavancin, and improved methods of treatment ofantibiotic-resistant bacterial infections. In particular, the inventionprovides dalbavancin compositions having activity against one or moreantibiotic resistant strains of bacteria, such as MRSA, which may beadministered in a dosing regimen of once every 5-7 days or longer.

Dalbavancin, which is also referred to in the scientific literature asBI 397 or VER001, is a semi-synthetic glycopeptide mixture, theproperties of which have been reported in U.S. Pat. Nos. 5,606,036,5,750,509, 5,843,679, and 5,935,238.

As used herein, the term “dalbavancin” refers to compositions comprisingone or more, preferably two or more, in some cases three or more, insome cases four or more, in some cases five or more closely relatedhomologs, termed “A₀,” “A₁,” “B₀,” “B₁,” “C₀,” “C₁,” “B₂” or “C₂,” “D₀”and “D₁,” as described below, or monomers, multimers (i.e., dimer orhigher order multimer), tautomers, esters, solvates, or pharmaceuticallyacceptable salts thereof. As used herein, “dimer” or “multimer” refersto either a homodimer or homomultimer, i.e., a dimer or multimercomposed of monomers of the same dalbavancin homolog, or a heterodimeror heteromultimer, i.e., a dimer or multimer composed of monomers of atleast two different dalbavancin homologs. The factors differ in thestructures of the fatty acid side chains of the N-acylaminoglucuronicacid moiety, with the exception of C₂ (a.k.a. B₂). Mass spectrometry ofthe C₂ component has indicated the presence of an additional methylenegroup on the terminal amino group. Dalbavancin often includes “MAG,” anon-homolog variant described below that lacks the acylglucuronaminemoiety. Individually, dalbavancin homologs and MAG are sometimesreferred to herein as “dalbavancin components.”

Dalbavancin is prepared by chemical modification of the naturalglycopeptide complex A-40,926 as described in Malabarba and Donadio(1999) Drugs of the Future 24(8):839-846. The predominant component ofdalbavancin is Factor B₀, which accounts for >75% of the whole complex.

The amount of each of the components present in a dalbavancincomposition is dictated by a variety of factors, including, for example,the fermentation conditions employed in the preparation of the naturalglycopeptide complex A-40926, which is the precursor to dalbavancin(see, e.g., U.S. Pat. No. 5,843,679), the conditions employed to recoverA-40926 from the fermentation broth, the chemical reactions employed toselectively esterify the carboxyl group of the sugar moiety of A-40926,the conditions employed to amidate the peptidyl carboxyl group, theconditions employed to saponify the ester of the carboxyl group of theN-acylaminoglucuronic acid function, the conditions employed to recoverdalbavancin from the synthetic mixture, and the like.

In preferred embodiments, dalbavancin compositions comprise at leastabout 80 to about 98% by weight of the B₀ component. In particularlypreferred embodiments, dalbavancin comprises the following amounts ofB₀: TABLE 1 Preferred Amounts of B₀ Component in Dalbavancin CompositionPreferred¹ More Preferred¹ Even More Preferred¹ 80-98 80-97 80-96 81-9881-97 81-96 82-98 82-97 82-96 83-98 83-97 83-96 84-98 84-97 84-96 85-9885-97 85-96 86-98 86-97 86-96 87-98 87-97 87-96 88-98 88-97 88-96 89-9889-97 89-96 90-98 90-97 90-96¹each range represents the mole % of B_(o) relative to the totaldalbavancin_components present in the dalbavancin composition includingMAG

In one embodiment, the dalbavancin composition or formulation contains aminimal amount, if any, of Components C₀, C₁, and C₂ (B₂). In anotherembodiment, the dalbavancin composition or formulation does not containany of Components C₀, C₁, and C₂ (B₂).

Individual dalbavancin factors have previously been purified by HPLC andcharacterized by NMR. In U.S. Pat. No. 5,750,509, Malabarba et al.described the antibiotic A 40926 derivative, which was characterized ashaving a carboxy, (C₁-C₄) alkoxy-carbonyl, aminocarbonyl, (C₁-C₄)alkylaminocarbonyl or hydroxymethyl substituent on theN-acylaminoglucuronyl moiety and a hydroxyl or a polyamine substituentin position 63 of the molecule. The compounds of the invention werefound to have high in vitro activity against glycopeptide resistantEnterococci and Staphylococci. Malabarba et al., however, neitherrecognized combinations of factors that were pharmaceutically beneficialnor identified or characterized the degradation product that lacks theacylglucuronamine moiety. Malabarba et al. never monitored for MAG orproduced a sterile form. Malabarba et al. only purified a small amountby HPLC and did not do quantitative mass analysis.

The chemical structure of several of the dalbavancin components isdepicted in Formula II below:

Dalbavancin Molecular Component R R₁ Weight A_(o) —CH(CH₃)₂ H 1802.7 A₁—CH₂CH₂CH₃ H 1802.7 B_(o) —CH₂CH(CH₃)₂ H 1816.7 B₁ —CH₂CH₂CH₂CH₃ H1816.7 B₂ or C₂ —CH₂CH(CH₃)₂ CH₃ 1830.74 C₀ —CH₂CH₂CH(CH₃)₂ H 1830.7 C₁—CH₂CH₂CH₂CH₂CH₃ H 1830.7 D₀ —(CH₃)₂ H 1788.66 D₁ —CH₂CH₃ H 1788.66 MAG— H 1459.27

All of the above dalbavancin components are bactericidally activeagainst a number of Gram-positive bacteria. However, one non-homologousdalbavancin component, termed “MAG,” which lacks an acylglucoronaminemoiety present in other components, is less bactericidally effective,both in vivo and in vitro, than other dalbavancin components. (SeeTables 2 and 3). MAG is thought to be a decomposition product of one ormore of the other dalbavancin components. Accordingly, in a preferredembodiment, the amount of MAG in dalbavancin is less than about 4, 3.5,3, 2.5, 2, 1.5, 1, or 0.5 mole percent of all dalbavancin componentspresent, including MAG. TABLE 2 ED₅₀s of MAG in comparison withdalbavancin and vancomycin against Staph. aureus murine septicaemias MICED₅₀ Microorganism Compound (μg/mL) (mg/kg) MSSA Staph. aureus Smith 819MAG 0.5 0.3 Dalbavancin 0.06 0.05 Vancomycin 0.5 1.7 MRSA Staph. aureus3817 MAG 0.5 1.6 Dalbavancin ≦0.03 0.7 Vancomycin 0.5 1.0** Treatment: once within 10 min from infection by sc route

TABLE 3 Microbiological Activity MIC μg/ml Microorganism MAG DalbavancinL 819 S aureus Smith 0.5-0.5-0.25 ≦0.13-0.06- <0.03 L 819 S aureus Smith50% 1-1-1 1-2-2 serum L 613 S aureus clin. isolate 1-0.5-0.50.5-0.06-<0.03 3797 S aureus clin. Isolate 0.5-0.5 2-2 GISA 3817 Saureus clin. Isolate 0.5 <0.03 L 147 S epidermidis 0.25-0.13-≦0.13-<0.03- 0.13 <0.03 L 49 S pyogenes C203 0.5-0.5-0.25 ≦0.13-<0.03-<0.03 L 44 S pneumoniae UC41 1-0.5-0.5 0.25-<0.03- <0.03 L 602 Shaemolyticus clin. >32-32 >32->32 isolate L 149 E faecalis 0.25-0.5-0.50.06-0.06 L 562 E faecalis clin. isolate 32->32->32 >32->32->32 L 1666E. faecium Van-A >32->32- >32->32->32 >32 L 102 B subtilis ATCC0.13-0.13 <0.03-<0.03 L 47 E coli SKF12140 >32->32- >32->32->32 >32 G16440 E coli iperperm 8-8-16 16-8-16 L 79 Pvulgaris >32->32- >32->32->32 >32 L 4 P aeruginosasATCC10145 >32->32- >32->32->32 >32 L 145 C albicansSKF2270 >32->32- >32->32->32 >32

Additional modifications in the final drug substance originate from thefermentation process, the subsequent chemical steps, or drying. IsoB₀(RRT 0.76) is a diastereomer of dalbavancin B₀ resulting fromepimerization of a labile proton in the dalbavancin core structure. Itoriginates during the deacetylation of the precursor A-40926 with base,forming the diastereomer of A-40,926. This diastereomer is subsequentlyconverted to IsoB₀ in the chemical modification steps that follow. IsoB₀is well resolved in the HPLC release method and is the only stereoisomerdetected in the API. Laboratory data has demonstrated that IsoB₀ is notformed during the basic hydrolysis procedure that converts the esterform (MA-A-1) to dalbavancin. IsoB₀ has been characterized by NMR and MSand has demonstrated in vivo biological activity that is comparable tothat of dalbavancin B₀.

The level of IsoB₀ in a dalbavancin composition is less that about 3.5%,alternatively less than about 3.0%, alternatively less than about 2.5%,alternatively less than about 2.0%, alternatively less than about 1.5%,alternatively less than about 1.0%, alternatively less than about 0.5%,alternatively less than about 0.4%, alternatively less than about 0.3%,alternatively less than about 0.2%, or alternatively less than about0.1%.

The level of IsoB₀ in a dalbavancin composition may alternatively bebetween about 0.5% ot about 3.0%, alternatively between about 0.5% toabout 2.8%, alternatively between about 0.5% to about 2.5%,alternatively between about 0.5% to about 2.3%, alternatively betweenabout 0.5% to about 2.0%, alternatively between about 0.5% to about1.8%, alternatively between about 0.5% to about 1.5%, alternativelybetween about 0.5% to about 1.3%, or alternatively between about 0.5% toabout 1.0%, or alternatively between about 0.1% to about 0.5%.

The level of IsoB₀ in a dalbavancin composition is alternatively notless than about 1.0%, alternatively not less than about 1.5%,alternatively not less than about 2.0%, alternatively not less thanabout 2.5%, alternatively not less than about 3.0%, alternatively notless than about 3.5%, alternatively not less than about 4.0%,alternatively not less than about 4.5%, or alternatively not less thanabout 5.0%.

Dalbavancin is thought to inhibit the biosynthesis of the bacterial cellwall by binding to D-alanyl-D-alanine-terminating precursors ofpeptidoglycans. Dimeric or higher order multimers of dalbavancin maypossess further antibacterial properties by interaction of thelipophilic side chains with the cytoplasmic membrane of bacteria. See,for example, Malabarba and Ciabatti, et al. (2001) Current MedicinalChemistry 8:1759-1773. A further elaboration on dalbavancin multimersmay be found in U.S. Ser. No. 10/714,166, entitled “DALBAVANCINCOMPOSITIONS FOR TREATMENT OF BACTERIAL INFECTIONS,” filed on Nov. 14,2003, as Attorney Docket No. 34231-20052.00, the disclosure of which ishereby incorporated by reference in its entirety.

In vitro, nonclinical, and clinical data indicate dalbavancin to be ofbenefit for the treatment of serious Gram-positive infections caused byMRSA and CoNS, and all streptococcal and non-VanA enterococcal species,including VanB and VanC phenotypes poorly susceptible or resistant tovancomycin.

Dalbavancin is more active in vitro against staphylococci (includingsome teicoplanin-resistant strains) than teicoplanin and vancomycin.Dalbavancin has better activity against streptococci, includingpenicillin-resistant strains, than teicoplanin or vancomycin.Dalbavancin is active in vitro and in vivo against a number ofGram-positive bacteria, including most drug resistant strains.

Dalbavancin is typically administered to an individual as a dalbavancincomposition. As used herein, the term “dalbavancin composition” or“dalbavancin formulation” refers to a composition, typically apharmaceutical composition comprising dalbavancin, as defined above, andone or more other non-dalbavancin components such as, for example, apharmaceutically acceptable carrier, a stabilizer, a buffers, or othersimilar components.

As shown in Example 1, dalbavancin is effective at dose intervals of oneweek. Thus, an advantage of dalbavancin versus other treatment optionsis the ability to administer this antibiotic on a once-weekly basis,thereby maximizing patient compliance and potentially minimizing theneed for or decreasing the length of a hospital stay for parenteralantibiotic administration. Less frequent dosing often permits treatmenton an out-patient basis, thus decreasing treatment costs. As furthershown in Example 1, a second dose of dalbavancin approximately one weekafter administration of the first dosage, where the second dose isapproximately one-half the first dose, unexpectedly provides significantimprovement in the efficacy of treatment.

Methods of Use

Previously, various dosing regimens, including single dose and multipledose regimens, for dalbavancin have been reported. Leighton et al.reported a multi-dose administration with an optimal dose ratio (loadingdose (LD)/maintenance dose (MD)) of 10:1. In this study, dose escalationproceeded to 1120 mg single dose (SD) and a multiple dose regimen up to500 mg BID Day I followed by 100 mg daily for 6 successive days.Leighton et al. “Dalbavancin: Phase I Single and Multiple-Dose PlaceboControlled Intravenous Safety, Pharmacokinetic Study in HealthyVolunteers,” 41^(st) ICAAC Abstracts, Chicago, Ill., Sep. 22-25 2001,Abstract No. 951, p. 25,

Leighton et al. also described other single and multiple doseadministrations. In the single dose studies, reported dose escalationproceeded via a series of 140 mg, 220 mg, 350 mg, 500 mg, 630 mg, 840mg, and 1120 mg. In the multiple dose phase, the dosing consisted of aloading dose, administered as two equal doses given 12 hours apart,followed by maintenance doses. The starting regimen was a loading doseof 150 mg BID followed by a maintenance dose of 30 mg per day for 6days. Dose escalation proceeded as follows: 200 mg BID/40 mg, 300 mgBID/60 mg; 400 mg BID/80 mg, and 500 mg BID/100 mg. Leighton et al.“Dalbavancin: Phase I Single and Multiple-dose Placebo ControlledIntravenous Safe Pharmacokinetic Study in Healthy Volunteers.” 41^(st)ICAAC, Chicago, Ill., December 2001, Poster No. 951.

White et al. reported dosing regimens of single 0.5 hour intravenousinfusion of 70 mg, 140 mg, 220 mg, or 360 mg. The multi-dose regimenconsisted of 70 mg administered daily for 7 days. White et al.“V-Glycopeptide: Phase 1 Single and Multiple-Dose Placebo ControlledIntravenous Safety, Pharmacokinetic, and Pharmacodynamic Study inHealthy Subjects.” 40^(th) ICAAC, Toronto, Canada, Sep. 17-20, 2000,Poster No. 2196 and Abstract No. 2196. All of the above-mentionedreferences are hereby expressly incorporated by reference in theirentirety.

Novel methods are provided for administration of dalbavancin to anindividual in need of treatment for a bacterial infection. Treatment caninclude prophylaxis, therapy, or cure. Methods include administration ofone or more unit doses of dalbavancin in a therapeutically orprophylactically effective amount.

As used herein, “therapeutically effective amount” refers to the amountof dalbavancin that will render a desired therapeutic outcome (e.g.,reduction or elimination of a bacterial infection). A therapeuticallyeffective amount may be administered in one or more doses. A“prophylactically effective amount” refers to an amount of dalbavancinsufficient to prevent or reduce severity of a future bacterial infectionwhen administered to an individual who is susceptible to and/or who maycontract a bacterial infection, e.g., by virtue of a medical procedureor stay in the hospital, or exposure to an individual with a bacterialinfection. Dalbavancin is generally administered in a pharmaceuticallyacceptable carrier.

Dalbavancin is often provided as a hydrochloride salt, which is freelysoluble in water.

Typically, dalbavancin is administered as a “unit dose” in a dalbavancinformulation which includes an amount of dalbavancin sufficient toprovide a therapeutically or prophylactically effective plasma level ofdalbavancin for several days, often at least about 5 days, one week, or10 days, when administered to an individual.

As used herein, “individual” refers to a vertebrate, typically a mammal,often a human.

All homologs of dalbavancin described above exhibit a prolongedhalf-life in plasma, often 9 days or more, although MAG is thought tohave a shorter half-life than other homologs. The long half-life permitslonger intervals between dosages than vancomycin or teicoplanin. Asdescribed in Example 1, weekly dosing of dalbavancin is effective forcontrol of bacterial infections, in contrast to the twice daily dosingschedule which is often used for vancomycin or the once daily schedulegenerally used for teicoplanin. Less frequent dosing of dalbavancinoffers significant treatment advantages over vancomycin and teicoplanin,particularly with regard to improved convenience and patient compliancewith the treatment regimen. Surprisingly high doses (i.e., resulting insurprising high and long-lasting serum levels) can be administered, andwith less frequency than other available treatment options. The noveldosage regimen available for dalbavancin results in improved efficacybecause at concentrations required to effect less frequent dosing,dalbavancin exhibits minimal adverse effects in vivo, evidencing a largepharmaceutical window, and further because blood levels of dalbavancinare maintained above minimum bactericidal levels for the entiretreatment protocol, evidencing a prolonged serum half-life fordalbavancin. The combination of the large pharmaceutical window coupledwith prolonged serum half-life permits less frequent dosing ofdalbavancin.

In addition, dalbavancin is preferably formulated with a stabilizerwhich inhibits degradation of one or more of the components ofdalbavancin. In one preferred embodiment, dalbavancin is formulated witha 1:2 weight ratio of mannitol:dalbavancin. In another preferredembodiment, dalbavancin is formulated with a 1:1:4 weight ratio ofmannitol:lactose:dalbavancin.

In some embodiments, a dalbavancin formulation is administered at adosage that results in therapeutically effective (i.e., bactericidal)plasma levels of the drug for several days, often at least about 5 toabout 10 days, often at least about one week. Generally, dalbavancin ismaintained in plasma at or above the minimum bactericidal concentrationof about 4 mg/l for at least 5 days. Often, dalbavancin is maintained ata plasma level of at least about 5 mg/l, often at least about 10 mg/l,often at least about 20 mg/l, often at least about 30 mg/l, often atleast about 40 mg/l, for at least 5 days, often at least about one weekor longer. Plasma levels of dalbavancin may be measured by methods thatare well known in the art, such as liquid chromatography, massspectrometry, or microbiological bioassay. An example of a method forquantitating dalbavancin in plasma is provided in Example 5.

Upper limits for dalbavancin plasma concentration levels are generallydictated by dosages which inhibit unacceptable adverse effects in thepatient population treated.

Dalbavancin compositions may be administered in a single dose or inmultiple doses. When administered as a single dose, the dalbavancincomposition is preferably formulated to contain sufficient amounts ofdalbavancin to effect antibacterial properties in vivo for at least 5days, preferably at least 7 days, and more preferably at least 10 days.

When multiple doses are employed, dalbavancin can be administered weeklyfor two or more weeks. In one embodiment, dalbavancin is administered inat least two doses, often in two doses about 5 to about 10 days apart,more often once a week for two weeks. As shown in Example 1, such adosing regimen provides significant advantages over conventionalantibiotic treatment protocols.

Dalbavancin compositions also may be administered in multiple doses twoor more days or at least one week apart or in one or more biweeklydoses. In some embodiments, a dalbavancin composition is administeredweekly, followed by biweekly, or monthly administration. In someembodiments, dalbavancin is administered at weekly intervals for 2, 3,4, 5, 6, or more weeks.

Most advantageously, daily dosing is not required because higher, lessfrequent doses are used. Single or multiple doses may range, forexample, from about 0.1 to about 5 grams. A single dose of about 0.1 toabout 4 grams, e.g., about 3 grams, may be administered for variousinfection treatments. Where multiple doses are administered, forexample, weekly, each dose may range, for example, from about 0.25 toabout 5.0 grams.

For embodiments in which a single dose is administered to treat aninfection, the amount of the dose may be, for example, about 0.1 toabout 5 grams, or about 0.5 to about 4 grams, or about 1 to about 3.5grams, or about 2 to about 3 grams e.g., about 3 grams. In someembodiments, a single dose of about 1, 1.5, 2, 2.5, or 3 grams isadministered for treatment of a bacterial infection. For embodiments inwhich a single dose is administered for prophylaxis, the amount of thedose may be, for example, about 0.1 to about 3 grams, or about 0.1 toabout 1 gram, e.g., about 0.5 or about 0.25 gram.

In dosing schemes that include multiple dosages, the individual dosagesmay be the same or different. In some embodiments, a first, higher doseis administered, that is, for example, about 1.5 to 10 times higher, incertain cases 9 times higher, in other cases 8 times higher, in othercases 7 times higher, in other cases 6 times higher, in other cases 5times higher, in other cases 4 times higher, in other cases 3 timeshigher, in other cases 2 times higher, than one or more subsequentdoses. For example, the first dose may be about 0.5 grams to about 5grams and the second dose about 0.25 grams to about 2.5 grams, the firstdose may be about 0.8 to about 2 g and the second dose about 0.4 toabout 1 gram, or the first dose may be about 0.4 to about 3 g and thesecond dose about 0.2 to 1.5 g.

In some embodiments, at least two dosages are administered wherein thefirst dosage includes about twice as much dalbavancin as subsequentdosages. In one embodiment, a first dosage includes about 1 gram ofdalbavancin and a subsequent dosage includes about 0.5 gram. In anotherembodiment, a first dosage includes about 0.5 gram of dalbavancin and asubsequent dosage includes about 0.25 gram.

In some embodiments, a dalbavancin composition is administered in twodoses of equal or different amount two or more days or at least aboutone week apart. Often, two doses of about 0.2 to about 1.5 grams ofdalbavancin are administered about 5 to about 10 days apart, more oftenabout 1 week apart. In one embodiment, a first dosage of about 1 gram ofdalbavancin and a second dosage of about 0.5 gram of dalbavancin areadministered about 1 week apart.

In a multiple dosing regimen, the time between doses may range, forexample, from about 5 to about 10 days, often about one week,alternatively about two weeks. Dose frequency may be, for example, twoweekly doses, or multiple weekly doses. The dosing interval, or timebetween doses, can be, for example, any of about 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or moredays. The number of doses given, can be, for example, one, two, three,four, five, six or more doses, each dose after the initial dose beinggiven after the selected dosage interval.

In a multiple dosing scheme, often the “trough level,” or the level ofdalbavancin in plasma after a first dose of dalbavancin and just priorto administration of a second dose, is at least about 4 mg/l.Preferably, the trough level at the end of a dosing interval such asabout one week is at least about 20 mg/l, more preferably at least about30 mg/l, and even more preferably at least about 40 mg/l.

Dalbavancin can be administered parenterally, e.g., intramuscularly(i.m.), intravenously (i.v.) (bolus or slow infusion), subcutaneously(s.c.), intraperitoneally (i.p.), or intrathecally (i.t.). The dosingschedule and actual dosage administered may vary depending on suchfactors as the nature and severity of the infection, the age, weight,and general health of the patient and the tolerance of a particularpatient to dalbavancin, but will be ascertainable to healthprofessionals. In one embodiment, a one gram intravenous dose ofdalbavancin is followed by a 0.5 gram intravenous dose one week later.

Administration and delivery of the drug to the patient, e.g.,intravenously, can be done at a controlled rate, so that theconcentration in the blood does not increase too quickly or causeprecipitation to occur. In some embodiments, dalbavancin is administeredat an appropriate rate such that the drug forms a complex withendogenous protein(s) in the bloodstream. Without intending to be boundto a particular theory, it is believed that endogenous protein, such ashuman serum albumin, can form a complex in vivo with one or twomolecules of dalbavancin homolog monomers. When a sufficient amount ofdalbavancin is present, it is believed that up to two molecules ofdalbavancin homolog will bind to the endogenous protein and it isfurther believed that this complex is formed by binding of separatehomolog molecules of dalbavancin at two different binding sites.Alternatively, it is possible that dimeric dalbavancin is binding to asingle binding site on the endogenous protein. A further elaboration onthe dalbavancin-endogenous protein complexes discussed above may befound in U.S. Ser. No. 10/713,924, entitled “COMPOSITIONS AND METHODSFOR TREATING BACTERIAL INFECTIONS WITH PROTEIN-DALBAVANCIN COMPLEXES,”filed on Nov. 14, 2003, as Attorney Docket No. 34231-20053.00, thedisclosure of which is hereby incorporated by reference in its entirety.

The infusion duration can be, for example, about 1 minute to about 2hours, or alternatively, from about 15 minutes to about 2 hours. Forexample, an infusion duration of about 30 minutes may be used where thedose is about 0.5 to about 1 gram. Intravenous administration undercontrolled rate conditions can generate concentrations of dalbavancin inthe body that are in great excess of what can be achieved in thesolution phase at physiological pH in vitro. Although not wishing to belimited by theory, this may be due to the formation of a complex ofdalbavancin with endogenous protein(s) such as serum albumin, which mayincrease the solubility of dalbavancin in plasma as compared to in vitrostudies.

Formation of a dalbavancin complex in vitro or ex vivo may permit fasteradministration, such as at least about 1 minute, at least about 10minutes or at least about 20 minutes. Such a complex can be achieved bymixing human serum albumin and/or another endogenous protein withdalbavancin, thereby forming the complex in vitro or ex vivo, and thenadministering this complex to the treated patient. Alternatively, thehuman serum albumin or other endogenous protein may be obtained fromautologous sources or by expression from a microorganism modified tocontain the gene for the protein.

The amount of dalbavancin administered may be any of the dosagesdisclosed herein. The dalbavancin dose is generally chosen such that thedrug will remain at a therapeutically or prophylactically effective(i.e., bactericidal) plasma level for an extended period of time, oftenat least 5 days, more often about one week or longer. Administration ofa dose of dalbavancin which produces and maintains bactericidalconcentrations for at least about one week (or about 5 to about 10 days)is preferred. A bactericidal concentration is defined as theconcentration of dalbavancin required to kill at least 99% of thebacteria present at the initiation of an in vitro experiment over a 24hour period. A minimum bactericidal concentration of dalbavancin inplasma is typically about 4 mg/l.

Examples of indications that can be treated include both complicated anduncomplicated skin and soft tissue infections (SSTI) (also known ascomplicated and uncomplicated skin and skin structure infections(SSSI)), blood stream infections (BSI), catheter-related blood streaminfections (CRBSI), osteomyelitis, prosthetic joint infections, surgicalprophylaxis, endocarditis, hospital or community acquired pneumonia,pneumococcal pneumonia, empiric treatment of febrile neutropenia, jointspace infections, and device infections (e.g., pace makers and internalcardiac defibrillators). Gram-positive or antibiotic-resistant bacterialinfections may be treated, such as a Staphylococcus, Streptococcus,Neisseria, or Clostridium genus infection, in particular Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus hemolyticus,Streptococcus pyogenes, Groups A and C Streptococcus, Neisseriagonorrhoeae, or Clostridium difficile.

The invention provides methods for treatment of skin and soft tissueinfections (SSTIs). Patients who may benefit from this treatment mayhave either deep or superficial infections. SSTI may involve deeper softtissue and/or require significant surgical intervention, such as forexample a major abscess, infected ulcer, major burn, or deep andextensive cellulitis. Infected surgical wounds may also be treated. Theeffectiveness of dalbavancin treating the skin is an unexpected andsurprising result because dalbavancin complexes with proteins in vivo(see Example 5).

The clinical presentation of skin and skin structure infection may varyfrom mild folliculitis to severe necrotizing fasciitis. The mode ofacquisition may also vary with community-acquired skin and skinstructure infections, which are often preceded by injuries resultingfrom occupational exposure or recreational activities, and are usuallyassociated with a greater diversity of pathogens. Hospital-acquired skinand skin structure infections are generally associated with surgicalprocedures, the development of pressure sores, and catheterization.Post-surgical infections are the third most frequent nosocomialinfection and account for 17% of all nosocomial infections reported tothe National Nosocomial Infection Surveillance System (NNIS). The mostfrequent source of infection is the patient's endogenous flora.Staphylococcus aureus, coagulase-negative staphylococci, andEnterococcus spp. are the pathogens most frequently isolated from SSTIs.

Symptoms of SSTI infections may include erythema, tenderness or pain,heat or localized warmth, drainage or discharge, swelling or induration,redness, or fluctuance. Patients that may benefit from treatment withthe methods of the invention include those with deep or complicatedinfections or infections that require surgical intervention, or patientswith underlying diabetes mellitus or peripheral vascular disease. Theseinfections are often caused by Gram-positive bacteria such asStaphylococcus or Streptococcus species, such as Staphylococcus aureusor Streptococcus pyogenes. Methods for treatment of a skin or softtissue bacterial infection include administering a therapeuticallyeffective amount of dalbavancin to an individual in need of treatment,in an amount and according to a dosing regime as discussed above. Insome embodiments, a dalbavancin composition is administeredintravenously in two doses, often about 5 to about 10 days apart, moreoften about 1 week apart. In some embodiments, the first dosage includesat least twice as much dalbavancin as the second dosage. In oneembodiment, the first dosage is about 1000 mg and the second dosage isabout 500 mg. In another embodiment, the first dose may be about 1200 mgand the second dose may be about 600 mg.

The invention also provides methods for prophylactic prevention of theonset of a bacterial infection, for example an infection caused byStaphylococcus aureus, or by a Neisseria or Clostridium genus bacterium.In a prophylactic method of the invention, a prophylactically effectiveamount of dalbavancin is administered to an individual who may besusceptible to contracting a bacterial infection, for example, through amedical procedure. Often, dalbavancin is administered in an amountsufficient to provide a prophylactically effective plasma level for atleast about 1 day, at least about 3 days, at least about 5 days, or atleast about one week or longer. Dalbavancin compositions may beadministered, for example, parenterally, e.g., via intramuscular (i.m.),intravenous (i.v.), intraperitoneal (i.p.), subcutaneous (s.c.), orintrathecal (i.t.) injection, prior or subsequent to surgery as apreventative step against infection. Dalbavancin compositions may beadministered immediately prior or subsequently to, 1 or more days orabout one week prior or subsequently to, or during an invasive medicalprocedure such as surgery or a stay in a medical care facility such as ahospital to prevent infection. A prophylactic method may be used in anysituation in which it is possible or likely that an individual maycontract a bacterial infection, including situations in which anindividual has been exposed to or is likely to be exposed to abacterially infected individual. For prophylactic methods, dalbavancincompositions may be administered as either a single dose or as two ormore doses of equal or different amount that are administered severaldays to about one week apart. In one embodiment, a dalbavancincomposition may be administered prior to or simultaneously withinsertion of an intravenous catheter in order to prevent a bloodstreamrelated infection.

For prophylactic methods, dalbavancin compositions may be administeredin a single dose or in multiple doses, according to any of the dosingschemes described above. Often, a dalbavancin composition isadministered as a single dose comprising about 0.1 to about 3 grams, orabout 0.1 to about 1 gram, e.g., about 0.25 gram or about 0.5 gram. Inone embodiment, a single dose of about 0.25 gram is administeredintravenously over a time frame of about 2 minutes to about 1 hour,e.g., about 30 minutes. In another embodiment, the dalbavancincomposition is administered intravenously simultaneously withadministration of another pharmaceutical (e.g., antibiotic) treatment.

In any of the therapeutic or prophylactic methods described above, thedalbavancin composition may be administered either simultaneously orsequentially with at least one other antibiotic. In some embodiments, atleast one other antibiotic that is effective (e.g., bactericidal)against one or more Gram-negative bacterial species and/or aGram-positive bacterial strain against which dalbavancin is noteffective is administered in addition to dalbavancin. In someembodiments, dalbavancin and at least one antibiotic that is effective(e.g., bactericidal) against at least one Gram-negative bacterialspecies is administered as a mixture in the dalbavancin composition.

Pharmacokinetics

Animal studies of dalbavancin were conducted using mice, rats, rabbits,dogs, and minipigs.

Various validated methods were used to quantify dalbavancin. DalbavancinAPI consists of 5 homologues (A₀, A₁, B₀, B₁, and B₂). Methods based onantibacterial activity measured all microbiologically active dalbavancincomponents. With chromatographic methods, the major component B₀ wasmeasured in combination with component B₁ (together 90% to 95% ofdalbavancin) and the result reported in terms of total dalbavancin.Liquid scintillation counting (LSC) and liquid chromatography withradiochemical detection (LC/RC) allowed for the measurement ofdalbavancin and metabolites in plasma, urine, and feces of animals givenradiolabeled drug. Whenever both dalbavancin and drug-derivedradioactivity were quantified in the plasma of animals given clinicallyrelevant doses, the concentration-time curves for drug and radioactivitywere superimposable. This indicates that virtually all of thedrug-derived radioactivity in plasma was intact drug.

The drug was widely distributed throughout the body into all testedorgans and tissues after IV dose administration. Dalbavancin penetratedinto the skin of rats and minipigs. Dalbavancin kinetics behaved in apredictable manner across species. Plasma clearance (CL) was found to beproportional to species body weight. Additionally, dalbavancin crossedthe rat placenta and was found in fetal rat plasma.

Dalbavancin is not a substrate, inhibitor, or inducer of hepaticcytochrome P450 isoenzymes. The drug is not metabolized in vitro by rat,dog, or human hepatic microsomes; rat, dog, or human hepatocytes; orhuman kidney microsomes. Dalbavancin did not affect the metabolism ofmarker substrates by isolated human hepatic microsomes. Administrationof dalbavancin to rats did not induce any P450 activity.

Similar dalbavancin metabolite profiles are observed across species. Two(2) metabolites (OH-dalbavancin and MAG) have been observed in the urineof rats, dogs, and humans. Both metabolites are either undetectable orclose to the limit of detection (<0.4 mg/L) in human plasma, have lessantibacterial activity than dalbavancin, and contribute little (ifanything) to in vivo activity. With clinically relevant doses, similarresults were generally observed in animal plasma.

The drug has dual routes (renal and fecal) of excretion and is excretedas intact drug and metabolites. Dalbavancin is excreted in the urine andfeces of rats, dogs, and humans. Most of the dose is excreted as intactdrug in the urine. OH-dalbavancin and MAG are found in the urine aswell. Some intact drug is found in animal feces along with tracequantities of metabolites. Human feces contained microbiologicallyactive dalbavancin components. Dalbavancin is also excreted into ratmilk. Therefore, dalbavancin is eliminated from the body via metabolism,urinary excretion, and fecal excretion. This would predict that littleif any dose adjustments would be required based on patient demographics.Suprisingly, patients with severe renal failure (creatinine clearanceCL_(CR) less than 30 mL/min) achieved higher levels than normal renalpatients (creatinine clearance CL_(CR) greater than 80 mL/min). Thesevere patients with a 1 g dose on Day 1 appeared similar to the levelsfor a normal patient who received 100 mg on Day 1 and 500 mg on Day 8.

Pharmaceutical Compositions

The invention provides pharmaceutical compositions formulated foradministration of dalbavancin according to the methods described above.Pharmaceutical compositions of the invention may be in the form of aunit dose of dalbavancin that includes an amount of dalbavancinsufficient to provide a therapeutically or prophylactically effectiveplasma level of dalbavancin for several days, often at least about 3days, at least about 5 days, or at least about one week or longer whenthe composition is administered to an individual, and a pharmaceuticallyacceptable carrier. Generally, a therapeutically or prophylacticallyeffective plasma level of dalbavancin is at least about 4 mg per literof plasma. Plasma levels of dalbavancin may be measured by well knownmethods in the art, such as those described above.

Dalbavancin may optionally be in a pharmaceutically acceptable form foradministration to an individual, optionally as a pharmaceuticallyacceptable, non-toxic salt.

Examples of suitable salts of dalbavancin include salts formed bystandard reaction with both organic and inorganic acids such as, forexample, hydrochloric, hydrobromic, sulfuric, phosphoric, acetic,trifluoroacetic, trichloroacetic, succinic, citric, ascorbic, lactic,maleic, glutamic, camphoric, glutaric, glycolic, phthalic, tartaric,lauric, stearic, salicylic, methanesulfonic, benzenesulfonic, sorbic,picric, benzoic, cinnamic, and the like acids. Representative examplesof bases that can form salts with dalbavancin include alkali metal oralkaline earth metal hydroxides such as sodium, potassium, calcium,magnesium, and barium hydroxide, ammonia and aliphatic, alicyclic, oraromatic organic amines such as methylamine, dimethylamine,diethylamine, ethanolamine, and picoline. (See, for example, U.S. Pat.No. 5,606,036.)

In some embodiments, a pharmaceutically acceptable aqueous formulationof dalbavancin is provided that is suitable for parenteraladministration, such as, for example, intravenous injection. Forpreparing such an aqueous formulation, methods well known in the art maybe used, and any pharmaceutically acceptable carriers, diluents,excipients, or other additives normally used in the art may be used. Inone embodiment, a pharmaceutically acceptable aqueous formulation forintravenous injection includes 5% dextrose.

Dalbavancin may be administered parenterally, i.e., the route ofadministration is by injection under or through one or more layers ofthe skin or mucous membranes. Since this route circumvents these highlyefficient protective barriers of the human body, exceptional purity of aparenteral dosage form free of microorganisms and insoluble particulatesmust be achieved. The process used in preparing such a dosage form mustembody good manufacturing practices that will produce and maintain therequired quality of the product in terms of sterility and therapeuticeffectiveness. In addition, the form should be stable when stored atroom temperature for a practical and convenient dosage form.

There are several conventional methods generally available forconverting bulk drug materials into a dosage form suitable forparenteral administration. These methods are generally outlined inRemington's Pharmaceutical Sciences, eighteenth edition, 1990(“Remington”).

Steam Sterilization

The USP defines steam sterilization as employing saturated steam underpressure for at least 15 minutes at a minimum of 121° C. in apressurized vessel. A drug in its solid form may be placed in anautoclave to affect the steam sterilization. A drug in its solution formmay be placed directly in an autoclave or contained in a sealedcontainer and placed in an autoclave to affect the same kind of steamsterilization.

Dry Heat Sterilization

In dry heat sterilization, a bulk drug material is subjected to elevatedtemperatures at relatively low humidity. Because dry heat is lessefficient than moist heat for sterilization, longer exposure times andhigher temperatures than those used in steam sterilization is required.The objective is to kill microorganisms by an oxidation process. Whileestablishing exact and correct time-temperature cycles is not routine,typical temperatures used are 140-170° C. from 1 to 3 hours.

Sterilization by Radiation

Sterilization by radiation may employ either electromagnetic radiationor particle radiation. Electromagnetic radiation, comprised of photonsof energy, includes ultraviolet, gamma, x-ray, and cosmic radiation.Gamma radiation, emitted from radioactive materials such as cobalt-60 orcesium-137, is the most frequently used source of electromagneticsterilization. The particle radiation most widely employed forsterilization is the beta particle or electron radiation.

Sterilizing Filtration

Sterilizing filtration is a process that removes, but does not destroy,microorganisms from a fluid stream. Such filtration is the method ofchoice for solutions that are unstable to other types of sterilizingprocesses.

Sterile Freeze-Drying (Lyophilization)

This method employs sterilizing filtration with the subsequent step ofseparating the sterilized drug from solution by sublimating the solutionafter the solution is frozen, leaving behind the drug substance. Themethod typically comprises the following steps:

-   -   1) dissolve bulk drug in aqueous solution    -   2) sterilize the solution by membrane filtration    -   3) fill the sterilized solution in opened, pre-sterilized vials        and place in freeze-drying chamber    -   4) freeze the solution in the vials    -   5) evacuate the chamber to sublime the ice under low temperature    -   6) increase the temperature to room temperature or above to        remove the residual water.        Sterile Freeze-Drying (Lyophilization) with Addition of Sterile        Water (Steam)

This method employs sterilizing filtration with the subsequent steps ofseparating the sterilized drug from the solution by freeze-drying(lyophilization), and adding sterile water in the form of steam.

Sterile Precipitation

This method employs sterilizing filtration with the subsequent step ofprecipitating the sterilized drug from solution. More specifically, abulk drug is first dissolved in water at an elevated temperature (aboveroom temperature), the heated solution is then filtered under asepticconditions to remove any microorganisms, and the filtered solution isthen cooled in order to precipitate the drug from solution. Theprecipitated drug is then separated from the solution by filtration orcentrifugation, and filled into containers by powder filling underaseptic conditions. For such powder filling to be practical, the drugmust have good flow properties—the powder should generally be granular,non-crystalline and of uniform particle size.

A pharmaceutical composition for parenteral administration includesdalbavancin and a physiologically acceptable diluent such as sterile ordeionized water, for injection, physiological saline, 5% dextrose, watermiscible solvent (e.g., ethyl alcohol, polyethylene glycol, propyleneglycol, etc.), non-aqueous vehicle (e.g., oil such as corn oil,cottonseed oil, peanut oil, and sesame oil), or other commonly useddiluent. The formulation may additionally include a solubilizing agentsuch as polyethylene glycol, polypropylene glycol, or other knownsolubilizing agent, buffers for stabilizing the solution (e.g.,citrates, acetates, and phosphates) and/or antioxidants (e.g., ascorbicacid or sodium bisulfite). (See, for example, U.S. Pat. No. 6,143,739.)Other suitable pharmaceutical carriers and their formulations aredescribed in “Remington's Pharmaceutical Sciences” by E. W. Martin. Asis known in the art, pharmaceutical preparations of the invention mayalso be prepared to contain acceptable levels of particulates (e.g.,particle-free) and to be non-pyrogenic (e.g., meeting the requirementsof an injectable in the U.S. Pharmacopeia).

In one embodiment, a pharmaceutical composition is provided bydissolving a dried (e.g., lyophilized) dose of dalbavancin, oftencontaining a stabilizer or mixture of stabilizers, in an amount of waterand preferably deionized water in a volume sufficient for dissolving thelyophile. Typically, the amount of water sufficient for dissolution isapproximately 10 mL and the resulting pH of the dalbavancin solution isabove 3.0, and about 3.5 to 4.5. This solution is further diluted in asecondary diluent, often containing 5% dextrose, such as an amountcontained in a drip bag for intravenous administration, raises the pH ofthe dalbavancin solution to about 5 to 5.5. In another embodiment, thepH of the dalbavancin solution in a drip bag is about 4.5. The secondarydiluent may be deionized and sterile water for injection. In oneembodiment, the aqueous diluent is 5% dextrose.

Pharmaceutical compositions for parenteral administration may be made upin sterile vials containing one or more unit doses of dalbavancin in atherapeutically or prophylactically effective amount as described above,optionally including an excipient, under conditions in whichbactericidal effectiveness of dalbavancin is retained. The compositionmay be in the form of a dry (e.g., lyophilized) powder. Prior to use,the powder is reconstituted with a physiologically acceptable diluentand then withdrawn via syringe for administration to a patient. Apharmaceutical formulation as described above may be sterilized by anyacceptable means including, for example, e-beam or gamma sterilizationmethods, or by sterile filtration.

A typical formulation for parenteral administration may includedalbavancin at a concentration such as about 0.1 to about 100 mg, about0.5 to about 50 mg, about 1 to about 10 mg, or about 2 to about 4 mg ofdalbavancin per ml of final preparation.

In some embodiments, a pharmaceutical composition in accordance with theinvention includes a mixture of dalbavancin and one or more additionalantibiotics. Preferably, at least one non-dalbavancin antibiotic in themixture is effective (e.g., bactericidal) against one or more species ofGram-negative bacteria, such as, for example, azthreonam, and/or againstone or more Gram-positive bacterial strains against which dalbavancin isnot effective, such as, for example, ilnezolide or daptomycin. Themixture may also include a pharmaceutically acceptable carrier asdescribed above.

In some embodiments, pharmaceutical compositions of the inventioninclude one or more stabilizing substances which inhibit degradation ofone or more of the components of dalbavancin to less active or inactivematerials, homologs, or related materials, for example, MAG. As usedherein, “stabilizing substance” or “stabilizer” refers to a substancethat stabilizes the level of one or more of the constituent componentsof dalbavancin, for example, B₀, in the composition. A “stabilizingeffective amount” refers to an amount of a stabilizer sufficient toenhance long-term stability of one or more components of a dalbavancincomposition. In some embodiments, a stabilizing effective amount may beprovided by a mixture of two or more stabilizing substances, each ofwhich alone is not present in an amount sufficient to provide astabilizing effect.

Examples of stabilizers include, for example, nonionic substances suchas sugars, e.g., mono-, di-, or polysaccharides, or derivatives thereof,sugar alcohols, or polyols. Such stabilizing substances include, forexample, mannitol, lactose, sucrose, sorbitol, glycerol, cellulose,trehalose, maltose, raffinose, dextrose, low and high molecular weightdextrans, or mixtures thereof.

In addition to sugars, stabilizers may also be amino acids. Amino acidstabilizers include natural and synthetic amino acids and amino acidderivatives. In a preferred embodiment, the amino acids are glycine,alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, andasparagine. Stabilizers may also be cyclodextrins, cyclic amides such asniacinamide and benzamide, salicylic acid, and its ortho-, meta-,para-substituted hydroxyl acids and esters.

In addition to adding stabilizers, adjusting the pH of the compounds wasfound to increase the stability of the composition. The particular pH ofthe composition that increases or maximizes the stability depends on thetype and amount of stabilizer added. The pH may be preferably about 1-7,more preferably 2-6, more preferably 3-5, more preferably 4-5, morepreferably approximately 4.5.

In one embodiment, the pharmaceutical composition includes a weightratio of 1:2 mannitol:dalbavancin. In another embodiment, thepharmaceutical composition includes a weight ratio of 1:1:4mannitol:lactose:dalbavancin. Surprisingly, it has been found that acombination of mannitol and lactose provides a greater stabilizingeffect than either substance alone. Often, the pH of a pharmaceuticalcomposition of the invention is, for example, about 3 to about 5, forexample about 3.5 or about 4.5.

In some embodiments, one or more procedures may be employed to reduceformation of MAG. For example, freeze drying of dalbavancin in thepresence of a stabilizing substance, such as mannitol and/or lactose,may be employed to reduce the amount of MAG formed.

Storage of dalbavancin compositions is often at lower than ambienttemperature, such as at about 5° C., to enhance stability.

Drying

Acetone removal from high molecular weight natural products can be quitedifficult because of it can form adducts with them. Its removal can beaccomplished in presence of water that replaces acetone in the solidproduct. Moreover, the process of drying dalbavancin is more delicatedue to the possibility of forming MAG, which depends on pH, temperature,vacuum, and time. In order to minimize MAG formation and speed up thedrying process, low endotoxin water is added or sprayed on the productduring the drying process to replace acetone. The excess water is laterreduced with additional hours of drying.

Drying is carried out under vacuum (internal temperature at <25° C.).When most of the acetone has been removed from the product, about 20%(w/w) of low endotoxin water is sprayed on the solid product and dryingis continued at the same conditions. To follow the drying process,acetone and Karl Fischer (K.F.) analyses are performed, and additionalwater is sprayed on the solid product until the residual acetone is lessthan 1.5%.

Study 25

Drying at 20-30° C. at 50 mbar.

This procedure was first simulated in the laboratory using a rotaryevaporator. Dalbavancin batch 027 was dried at 30° C. without addingwater giving a product which contained 6.4% of MAG and 3.2% of acetone.126 g of this dried dalbavancin batch (batch 027, HCl/Dalba ratio 1.7mols/mol) was charged in a 1 L round bottomed flask and the solid wassprayed with 25 mL of water every two hours, maintaining the temperatureof the water bath at 20-30° C. and the vacuum at 50 mbar. Samples of thedalbavancin were taken at specific times and analyzed for acetone, K.F.,and MAG analyses. The data obtained are reported in Table 4A. All of thepercentages of MAG reported in this table are the difference between theactual amount and the starting amount. TABLE 4A T P Delta water Time h °C. mbar water % Acetone % MAG % sprayed % 0 — — 1.6 3.2 — 20 2 20-25 508.3 2.8 0 20 4 20-25 50 15.6 1.9 0 20 6 20-25 50 23.3 0.9 0 — 8 30 5022.6 0.56 — — 10 30 50 21.8 0.43 0 — 12 30 50 20.8 0.22 0 —This experiment was completed with some additional hours of drying atthe same conditions, reducing the water content below 15%.Drying at 30° C. at 10 mbar.

125 g of Dalbavancin batch 027, MAG 6.4% (HPLC area %), was dried at 30°C. and 10 mbar for 24 hours by using the same equipment of the previousexperiment. The results obtained are reported in Table 4B. TABLE 4BDelta water Time h T ° C. P mbar KF % Acetone % MAG % sprayed % 0 — —1.6 3.21 — 20 2 30 10 13.0 1.09 0 20 4 30 10 15.08 1.18 0 20 6 30 1016.8 0.02 0 — 24 30 10 8.89 0.02 0

From these laboratory experiments, it can be concluded that the dryingprocess can be simplified by adding water to the solid product. Theseresults were unexpected since the degradation of dalbavancin to MAG is ahydrolysis step, one would expect that the increase in water wouldincrease the degradation process. In this way, acetone is rapidlyremoved from the product, thereby reducing the drying time and avoidingthe formation of additional MAG. Moreover, it can be observed (Table 4B)that a lower residual pressure gave a significant reduction of thedrying process. It is also surprising that the acetone level was reducedin the presence of a large amount of water.

Drying of Dalbavancin in Tumble Drier

Wet dalbavancin was put into a tumble drier, DR 216, and dried at 29-32°C. (external temperature) with maximum vacuum (<50 mbar). At thebeginning, the internal temperature decreased from 30 to 17-18° C. andthen, when it began to increase, a sample of product was taken foranalysis (water 17.2% and acetone 9.5%). At this point, low endotoxinwater (about 20%, w/w) was sprayed into the drier on the stirred productand additional samples were taken on time for analysis. After two hours,low endotoxin water was sprayed again until the amount of acetone waslower than 0.5%, (Table 4C). TABLE 4C External temperature 29-30° C.Pressure = 10-15 mbar Water MAG Time weight Int. added water Acetonearea h Kg Temp. mL (*) analysis # % % % 0 19.1 0.11 1 17.4 ** 2 18.3 **3 23.5 200201019 17.27 9.52 ** 4.5 26.8 500 ″ 13.59 4.10 0.13 6.5 28.1500 ″ 19.60 0.61 0.16 8.5 28.1 ″ 22.38 0.13 0.12 9.5 28.5 ″ 19.86 0.120.13 11.5 21.9 ″ 17.68 0.09 0.31 15 29.5 ″ 14.11 n.d. 0.38Final weight 2592 g, An. # 200201027: water(K.F.) 13.93%, acetone 0.12%,MAG 0.38%.(*) water was sprayed in the tumble drier after samplingComparison of Drying in the Presence and Absence of Water

This study is a comparison between dalbavancin batch 027 and 027/Rdrying processes carried out in the absence (batch 027) and in thepresence of water (027/R). Dalbavancin batches 027 (14.6 Kg) and 027/R(8.3 Kg) were dried under vacuum at 25-35° C. (internal temperature)using a tumble drier (DR 216). Batch 027 was dried without adding water.In contrast, four additions of 400 mL each of low endotoxin water weresprayed on batch 027/R. During each drying, samples of dalbavancin weretaken at specific times for analysis. The results are reported in Table5. For batch 027, MAG was only determined at the end of the dryingprocess. TABLE 5 Lot 027/R Lot 027 Water temp. Press KF Acetone MAGtemp. Press KF Acetone MAG added hours (° C.) mbar (%) (%) (%) (° C.)mbar (%) (%) (%) (mL) 0 32 280 0.52 f (*) 30 171 0.22 2 29 32 (°) 3 — —23 57 16 6.7 0.23 7 — — 29 1 7.4 3.94 0.44 400 9 — — 28.4 2 8 2.6 0.48400 11 — — 28 4 10 1.5 0.59 400 12.5 — — 27 12 13.9 1 0.74 400 15 — —27.3 9 12 0.45 0.88 — 17 35 0 5.5 2.9 18 — — — — 27.5 8 11.5 0.26 0.96 —26 37.8 — — — 65 35.7 0 1.6 3.1 n.a. Analysis # 200101408 Analysis #200101944 MAG. 6.46% MAG 1.81% K.F 1.63% KF 11.86%  Acetone 3.14%Acetone 0.27%(*) An. # 200101392(°) An. # 200101913Full Scale Production Data

Below in Table 6 is a summary of the process and MAG levels between thefull scale campaigns of 2002 and 2004. TABLE 6 Campaign 2002 2004 Lot020004/R 020005/R 020006 DA204001 DA2040002 DA2040003 % MAG 2.3 0.8 1.70.4 0.3 0.3

In the 2002 Process, the dalbavancin product is dried under vacuum atless than 30° C. At various times, low endotoxin water is sprayed on theproduct. Drying is continued until the acetone content is less than0.5%.

In the 2004 process, dalbavancin API is recovered by centrifugation,washed with cold acetone (0-10° C.), and dried under vacuum (internaltemperature at <25° C.). MAG is the primary degradant of dalbavancin andits formation is dependent on temperature. During the drying process,water is added in order to displace the acetone to ultimately achieve anacetone content of less than 1.5% (GC). As apparent from Table 5, dryingat low temperature in the presence of water led to an unexpectedly loweramount of acetone present (compare 0.27% in Lot 027/R to 3.14% in Lot027) and an unexpectedly low amount of MAG degradation product (compare1.81% in Lot 027/R to 6.46% in Lot 027).

Therefore, a method for drying dalbavancin includes the steps of addingwater to the dalbavancin product in order to displace the acetone. Apreferred amount of water to be added is about 20% of the assumed finaldry product. Alternatively, the amount of water may be about 15%,alternatively about 25%, alternatively about 30%, alternatively about40%, alternatively about 45%, alternatively about 50% of the assumedfinal dry product.

A method for drying dalbavancin may include the steps of drying thedalbavancin product for about 2 hours without the addition of any waterat about 100 torr and at a temperature of less than about 25° C. Lowendotoxin water is then sprayed on the product and the product is thendried for about two more hours. The level of acetone is then sampled andchecked. If the level is more than about 1.5%, the additional water issprayed on the product and the product is again dried until the acetonelevel is less than about 1.5%.

Manufacture

All bulk solution manufacturing operations take place in a class100,000. (Grade D) area. Aseptic filling takes place in a class 100(Grade A) laminar airflow area.

A suitable manufacturing vessel is charged with about 80% of the Waterfor Injections theoretical batch volume. The solution is mixed and thetemperature is maintained between 15-30° C. The dalbavancin is added andmixed until it is dissolved. At least one stabilizer is added to thesolution and mixed until dissolved. Water for injection is added tobring the solution up to final volume and the pH is adjusted, ifnecessary, with either 0.1N HCl or 1.0N NaOH to an appropriate pH. Thebulk solution is sterilized by filtration through two 0.2 micronsterilizing filters in series into a sterilized receiving vessel. (Aprefilter can be used if necessary to aid in filter clarity or to reduceparticle load to the sterilizing filters). The solution is asepticallyfilled into sterile/depyrogenated Type I glass vials. Sterilesiliconised lyophilisation stoppers are partially inserted to thelyophilisation position and the vials are transferred to thelyophilisation chamber.

The lyophilisation process is monitored by the use of thermocoupleprobes for representative vials. Vials are frozen at −45° C. and heldfor 3 hours, after which vacuum is applied. The shelf temperature isadjusted to −25° C. When all thermocouples are −29° C. or warmer theshelf temperature is adjusted to 0° C. When all thermocouples are −5° C.or warmer the shelf temperature is adjusted to +30° C. When allthermocouples are +27° C. or warmer the vials are held for 14±2 hours.The chamber is restored to atmospheric pressure by the introduction ofsterile nitrogen which has been filtered through a 0.2 micron filter andthe vials are then sealed by collapsing the lyophilisation shelves.Vials are then removed from the chamber and the aluminum seals areapplied

All components and equipment are sterilized by appropriate processes.Vials are washed and sterilized in a hot air oven at a temperature notless than 255° C. for not less than 3 hours. Stoppers are steamautoclaved at a temperature of 123-125° C. and a chamber pressure ofabout 33 psi. The dwell time in the sterile range is typically 50-60minutes.

Validation of sterilization processes uses the “overkill” approach forboth steam and dry heat sterilization cycles. All sterilization cyclesprovide a sufficient lethality to provide at least a 10⁻⁶ probability ofmicrobial survival regardless of the naturally occurring microorganisms.All cycles are designed with lethalities sufficient to provide not lessthan 12 log reductions. Dry heat cycles will provide a minimum of a 3log endotoxin reduction.

Stability Studies

Dalbavancin was found to decompose during the freeze drying process. Theaddition of a stabilizer was found to decrease the amount ofdecomposition of the active component B₀ during the stability studies.

The stability of various lyophilized formulations of dalbavancin at 25°C. and 40° C. over time is shown in FIGS. 1-4. Under FDA guidelines, aproduct that is stable for three to six months at 40° C. is assumed tobe stable for two years at room temperature. FIGS. 1A, 2A, 3A, and 4Ashow the decrease in amount of dalbavancin component B₀, which is one ofthe bactericidally active dalbavancin components. As discussed above,component B₀ is also one of the major components of most dalbavancincompositions. FIGS. 1B, 2B, 3B, and 4B show the increase in the amountof MAG, a less active component thought to be a decomposition product ofone or more of the other dalbavancin components. Table 7 lists thecompositions for each of the formulations used in the stability studies,the results of which are shown in FIGS. 1-4. Any of these compositionscould be used to produce a stable, sterile, particle free dosage form.TABLE 7 Compositions of Various Dalbavancin Formulations DalbavancinMannitol Lactose Composition (mg/vial) (mg/vial) (mg/vial) pH A 250 62.5— 3.4 B 250 — — 3.69 C 250 62.5 — 3.80 D 250 — — 3.01 E 250 62.5 — 3.01F 250 — — 4.5 G 250 62.5 — 4.5 H 250 62.5 — 5.3 I 250 125 — 5.0 J 25062.5 — 5.0 K 250 125 — 4.5 L 250 62.5 — 4.5 M 250 62.5 62.5 4.5 N 250 —125 4.5 O 250 125 — 3.3

As seen in FIGS. 1B and 2B, at T=0, there is already a significantamount (greater than 4%) of MAG present for Composition D, whichcontains dalbavancin with no other non-dalbavancin components and whichhas not been pH adjusted (pH about 3.01), at 25° C. and 40° C.,respectively. At the higher temperature, the formation of MAG increasedat a far greater rate. After 3 and 6 months at 40° C., Composition D had21.0% MAG and 23.7% MAG, respectively (see FIG. 2B). This implies thatpure dalbavancin is highly unstable, and that merely freeze-drying thedalbavancin results in significant degradation. In addition, normaldrying also results in formation of the MAG degradation product. Storageat −20° C. is required for some formulations of dalbavancin.

When the pH is increased, without the addition of any othernon-dalbavancin components, the stability increased. Composition D wasnot pH adjusted and had a pH of about 3.01. Composition B was adjustedto pH 3.69. Composition F was adjusted to pH 4.5. As seen in FIGS. 1Aand 2A, as the pH was increased, there was less initial degradation ofB₀ and less overall degradation over time. Analogously, in FIGS. 1B and2B, there was less MAG formation in the compositions that were adjustedto a higher pH, both initially and over time at both temperatures.

The addition of mannitol was also shown to increase the stability ofdalbavancin significantly. The degradation of factor B₀ and increase inMAG over time was also reduced significantly in comparison withComposition D, which was also not pH adjusted but contained no mannitol.As seen in Composition E (62.5 mg mannitol, about pH 3.01), even withoutany pH adjustment, there was significant improvement in stability, bothin the initial freeze-drying process and over time. At time T=0, thereis approximately 2% of MAG present at T=25° C. (see FIG. 1A) and T=40°C. (see FIG. 2A), which is less than half the amount of MAG present inComposition D at T=0.

As the pH was increased and the amount of mannitol was held constant,the stability of the dalbavancin also increased. A comparison ofCompositions E, C, and G illustrate that, as the pH was increased fromabout 3.01 to 3.8 to 4.5, the amount of degradation of dalbavancin alsodecreased. As seen in FIGS. 1A and 2A, as the pH was increased, therewas less initial degradation of B₀ and less overall degradation overtime. Analogously, in FIGS. 1B and 2B, there was less MAG formation inthe compositions that were adjusted to a higher pH, both initially andover time.

Increasing the amount of mannitol, while keeping the pH constant, alsoresulted in an increase in stability. For instance, although Compounds Land K, which contain 62.5 mg and 125 mg of mannitol at pH 4.5,respectively, have similar amounts of B₀ at T=0, the change in thepercentage of B₀ after 12 months was significantly less for Compound K.A similar pattern can be seen for Compound J and I, which contain 62.5mg and 125 mg of mannitol at pH 5.0, respectively.

Although changing the pH of the compositions containing only mannitoldid result in changes in the amount of degradation of B₀, there was nopredictable trend. Compounds L, J, and H contain 62.5 mg of mannitol atpH 4.5, 5.0, and 5.3, respectively. The changes in the percentage of B₀after 12 months was 1.0, 1.2, and 0.7, respectively, at 25° C. Compounds0, K, and I contain 125 mg of mannitol at pH 3.3, 4.5, and 5.0,respectively. The changes in the percentage of B₀ for these compositionsafter 12 months was 0.5, 0.1, and 0.3, respectively, at 25° C. Notably,after 2 months at 40° C., the amount of component B₀ only decreased by1.9% in Compound O and 1.0% in Compound M (See FIG. 4A).

Although the change in the percentage of B₀ for Compound O (125 mg ofmannitol, pH 3.3) is similar to the other differences found for thecompositions containing 125 mg of mannitol (see Compositions I and K),as seen in FIG. 3A, the amount of initial degradation of B₀ at T=0 issignificantly less for Compound O (88.3% B₀). This is especially thecase when compared with Compound K (pH 4.5) and Compound I (pH 5.0),which had 85.3% B₀ and 85.5% B₀, respectively. The differences betweenthe amount of B₀ present and the amount of MAG present in Compound O(see FIGS. 3A and 3B) can most likely be explained by the fact that, asexplained previously, B₀ is not the only dalbavancin component thatdegrades to form MAG.

Lactose also appears to be a suitable stabilizer for dalbavancin. ForCompound N, which contains 125 mg of lactose at pH 4.5, the change inpercentage of B₀ over 12 months was only 0.6 at 25° C. After 2 months at40° C., the change in percentage of B₀ was only 1.4. Lactose alone,however, does not appear to stabilize the dalbavancin as well asmannitol. At pH 4.5, the B₀ component of Compound K (125 mg of mannitol)only decreased by 1.0 after 2 months at 40° C.

The combination of mannitol and lactose also appears to stabilizedalbavancin and is particularly preferred. Mannitol and lactose havesimilar stabilizing properties. Mannitol, however, is a diuretic.Therefore, in a preferred embodiment, the amount of mannitol isminimized. Compound M contains 62.5 mg each of mannitol and lactose. Asseen in FIG. 3A, the change in percentage of MAG over 12 months at 25°C. was only 0.6. In addition, as seen in FIG. 4A, the amount of MAG onlyincreased by 2.1% after 3 months at 40° C. This is less degradation thanthat seen for Compounds N (125 mg lactose) and K (125 mg mannitol),which both showed an increase in the amount of MAG of 2.9% after 3months at 40° C. It was unexpected that a combination of half of each ofthe amounts of mannitol and lactose used in other formulations wouldlead to a greater increase in dalbavancin stability.

Additional stability data for dalbavancin stabilized with mannitol andlactose at approximately pH 4.5 is reported in Tables 8A-D below forvarious temperatures. TABLE 8A Stability of Dalbavancin for InjectionLot 554399 at 40° C./75% TH (500 mg vial, mannitol and lactose, pH 4.5)TIME (MONTHS) TEST 0 1 3 6 Physical Appearance White to White to Whiteto White to off white to off white to off white to off white to paleyellow pale yellow pale yellow pale yellow solid solid solid solid HPLCAssay 95.4 95.7 92.2 91.6 Component Distribution (A₀ + A₁) 2.6 2.5 2.42.5 B₀ 87.0 86.0 85.4 84.9 (B₁ + B₂) 5.8 6.2 6.3 6.4 Degradants MAG 1.01.5 1.9 2.2 Maximum Individual Unspecified n.d. n.d. n.d. n.d. TotalDegradants 1.0 1.5 1.9 2.2 Reconstitution Time 115 57 47 55 Appearanceof Solution Clear to Clear to Clear to Clear to pale yellow pale yellowpale yellow pale yellow solution solution solution solution ParticulateMatter Particles ≧10 microns Meets — — — USP Particles ≧25 microns Meets— — — USP Moisture 0.4 0.5 0.5 0.6 pH of Reconstituted Solution 4.3 4.64.5 4.5 Sterility Meets — — — USPn.d. not detected— not tested at this time point, per stability protocol

TABLE 8B Stability of Dalbavancin for Injection Lot 554399 at 30° C./65%RH (500 mg vial, mannitol and lactose, pH 4.5) TIME (MONTHS) TEST 0 1 36 9 12 Physical Appearance White to White to White to White to White toWhite to off off off off off off white to white to white to white towhite to white to pale pale pale pale pale pale yellow yellow yellowyellow yellow yellow solid solid solid solid solid solid HPLC Assay 95.497.3 94.2 93.7 94.0 93.8 Component Distribution (A₀ + A₁) 2.6 2.5 2.52.5 2.4 2.4 B₀ 87.0 86.2 85.8 85.3 86.7 85.9 (B₁ + B₂) 5.8 6.2 6.4 6.55.5 6.4 Degradants MAG 1.0 1.1 1.3 1.5 1.7 1.7 Maximum IndividualUnspecified n.d. n.d. n.d. n.d. 0.1 n.d. Total Degradants 1.0 1.1 1.31.5 1.8 1.7 Reconstitution Time 115 146 53 40 37 49 Appearance ofSolution Clear to Clear to Clear to Clear to Clear to Clear to pale palepale pale pale pale yellow yellow yellow yellow yellow yellow solutionsolution solution solution solution solution Particulate MatterParticles ≧10 microns Meets — — — — — USP Particles ≧25 microns Meets —— — — — USP Moisture 0.4 0.4 0.4 0.5 0.6 0.5 pH of ReconstitutedSolution 4.3 4.5 4.5 4.4 4.5 4.5 Sterility Meets — — — — — USPn.d. Not detected— Not tested at this time point, per stability protocol.

TABLE 8C Stability of Dalbavancin for Injection Lot 554399 at 25° C./60%RH (500 mg vial, mannitol and lactose, pH 4.5) TIME (MONTHS) TEST 0 3 69 12 Physical Appearance White White White White White to off to off tooff to off to off white to white to white to white to white to pale palepale pale pale yellow yellow yellow yellow yellow solid solid solidsolid solid HPLC Assay 95.4 94.6 93.2 93.9 92.6 Component Distribution(A₀ + A₁) 2.6 2.5 2.5 2.5 2.4 B₀ 87.0 86.0 85.9 87.0 86.2 (B₁ + B₂) 5.86.4 6.3 5.7 6.4 Degradants MAG 1.0 1.2 1.2 1.4 1.4 Maximum Individualn.d. n.d. n.d. n.d. n.d. Unspecified Total Degradants 1.0 1.2 1.2 1.41.4 Reconstitution Time 115 55 68 54 50 Appearance of Solution Clear toClear to Clear to Clear to Clear to pale pale pale pale pale yellowyellow yellow yellow yellow solution solution solution solution solutionParticulate Matter Particles ≧10 microns Meets — — — Meets USP USPParticles ≧25 microns Meets — — — Meets USP USP Moisture 0.4 0.3 0.4 0.40.5 pH of Reconstituted 4.3 4.5 4.5 4.6 4.6 Solution Sterility Meets — —— Meets USP USPn.d. Not detected— Not tested at this time point, per stability protocol.

TABLE 8D Stability of Dalbavancin for Injection Lot 554399 at 5° C. (500mg vial, mannitol and lactose, pH 4.5) TIME (MONTHS) TEST 0 3 6 9 12Physical Appearance White White White White White to off to off to offto off to off white to white to white to white to white to pale palepale pale pale yellow yellow yellow yellow yellow solid solid solidsolid solid HPLC Assay 95.4 94.7 92.7 93.7 93.4 Component Distribution(A₀ + A₁) 2.6 2.5 2.5 2.5 2.4 B₀ 87.0 86.2 86.1 87.6 86.9 (B₁ + B₂) 5.86.4 6.3 5.7 6.4 Degradants MAG 1.0 0.9 0.9 1.0 0.9 Maximum Individualn.d. n.d. n.d. n.d. n.d. Unspecified Total Degradants 1.0 0.9 0.9 1.00.9 Reconstitution Time 115 90 88 75 72 Appearance of Solution Clear toClear to Clear to Clear to Clear to pale pale pale pale pale yellowyellow yellow yellow yellow solution solution solution solution solutionParticulate Matter Particles ≧10 microns Meets — — — Meets USP USPParticles ≧25 microns Meets — — — Meets USP USP Moisture 0.4 0.3 0.3 0.40.4 pH of Reconstituted 4.3 4.5 4.5 4.5 4.6 Solution Sterility Meets — —— Meets USP USPn.d. Not detected— Not tested at this time point, per stability protocol.

Data from further long-term stability studies are listed in Tables 9-11.Samples were analyzed by reverse-phase HPLC utilizing binary mobilephase gradient and a UV detection system to determine the stability ofthe dalbavancin formulation. Dalbavancin content was determined using anexternal reference standard. The percentage distribution of dalbavancincomponents was calculated by comparing the area of each single componentto the total area of all of the major drug components. The percentagedistribution of impurities was calculated by comparing each the area ofindividual impurity to the total chromatographic area.

Samples were tested for HPLC assay, bioassay, water content, microbiallimits, pH, HPLC distribution, or total impurities. The results areshown in Tables 9-11. Stability data for Batch 025 is shown in Tables9A, 10A, and 11A, which has 1.5% MAG at T=0. Stability data for Batch020005/R is shown in Tables 9B, 10B, and 11B, which has 0.8% MAG at T=0,as a result of a different drying process described that is describedbelow.

Dalbavancin demonstrated good stability during storage at −20° C. (seeTables 9A-B) with no significant changes in bioassay, HPLC assay, HPLCdistribution, or total impurities. The pH remained unchanged and aslight increase in water content was observed.

Degradation was observed over 36 months at 5° C. (see Table 10A) asevidenced by an increase in MAG content by about 2.9% and acorresponding decrease in Factor B₀ by about 3.6%. No trend in totalimpurities was observed and bioassay and pH remain essentiallyunchanged. Similar results are seen for Batch 020005 (see Table 10B).

More extensive degradation was observed during storage at 25° C. over 12months (see Table 11A) with an increase in MAG of about 10.2% and adecrease in factor B₀ of approximately 8.5%. Levels of other factors,related substances, and pH remained unchanged. The water contentincreased by about 1.6%. TABLE 9A Stability Data for UnformulatedDalbavancin Batch 025 at −20° C. (pH 3.5) TIME (MONTHS) TEST 0 3 6 9 1218 24 36 Physical Appearance — — — — — — — — HPLC Assay (anhydrous, 92.093.3 90.1 88.6 88.4 91.1 89.6 90.3 free base, % w/w) ComponentDistribution (HPLC % AUC) A₀ + A₁ 4.0 4.0 4.0 4.0 3.9 3.9 3.9 3.8 B₀83.4 83.5 82.1 82.9 84.4 83.8 83.5 82.7 B₁ + B₂ 7.5 7.4 8.1 8.0 7.4 7.37.5 7.9 Related Substances (HPLC % AUC) MAG 1.5 1.5 1.3 1.6 1.3 1.5 1.41.2 Maximum Individual 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Unspecified TotalImpurities 5.1 5.1 5.8 5.2 4.3 4.9 5.2 5.6 Moisture (% w/w) 11.0 13.312.1 12.6 12.1 12.8 12.2 14.2 pH 3.0 2.9 3.0 2.9 2.9 2.9 2.9 2.8Microbial Limits* <10, <10 cfu/g — — — — — — —— Not tested at this time point, per stability protocol.*Total aerobic count, total combined molds and yeast count

TABLE 9B Stability Data for Unformulated Dalbavancin Batch 020005/R at−20° C. TIME (MONTHS) TEST 0 3 6 9 12 18 Physical Appearance White WhiteWhite White White White to tan to tan to tan to tan to tan to tan powderpowder powder powder powder powder HPLC Assay (anhydrous, free base, %92.0 94.4 92.5 94.6 91.0 92.1 w/w) Component Distribution (HPLC % AUC)A₀ + A₁ 2.5 2.5 2.5 2.5 2.4 2.5 B₀ 87.1 87.5 86.7 86.6 86.5 87.3 B₁ + B₂6.2 5.6 6.2 6.2 6.6 5.9 Related Substances (HPLC % AUC) MAG 0.8 0.9 0.90.8 0.9 0.9 Maximum Individual Unspecified 0.2 0.2 0.2 0.3 0.2 0.2 TotalImpurities 4.3 4.5 4.6 4.7 4.4 4.4 Moisture (% w/w) 18.2 20.1 20.1 20.320.0 19.6 pH 2.4 2.5 2.4 2.5 2.5 2.6 Microbial Limits* 2.1 cfu/g — — — ——— Not tested at this time point, per stability protocol.*Total aerobic count, total combined molds and yeast count

TABLE 10A Stability Data for Unformulated Dalbavancin Batch 025 at 5°C.) TIME (MONTHS) TEST 0 3 6 9 12 18 24 36 Physical Appearance — — — — —— — — HPLC Assay (anhydrous, 92.0 91.0 90.3 91.8 90.5 89.3 88.2 87.3free base, % w/w) Component Distribution (HPLC % AUC) A₀ + A₁ 4.0 4.03.9 3.9 3.8 3.9 3.8 3.7 B₀ 83.4 83.1 81.3 81.8 83.3 82.2 81.3 79.8 B₁ +B₂ 7.5 7.4 7.9 7.7 7.2 7.2 7.2 7.5 Related Substances (HPLC % AUC) MAG1.5 1.8 2.0 2.5 2.8 3.3 3.8 4.4 Maximum Individual 0.2 0.3 0.3 0.2 0.20.2 0.2 0.3 Unspecified Total Impurities 5.1 5.5 6.9 6.5 5.7 6.7 7.7 9.0Moisture (% w/w) 11.0 12.3 11.8 13.2 15.0 14.8 14.9 17.6 pH 3.0 3.0 3.02.9 2.9 2.9 2.9 2.8 Microbial Limits* <10, <10 cfu/g — — — <1, 3 cfu/g —<1.1 cfu/g <1, <1 cfu/g— Not tested at this time point, per stability protocol.*Total aerobic count, total combined molds and yeast count

TABLE 10B Stability Data for Unformulated Dalbavancin Batch 020005/R at5° C. TIME (MONTHS) TEST 0 3 6 9 12 18 Physical Appearance White toWhite to White to White to White to White to tan tan tan tan tan tanpowder powder powder powder powder powder HPLC Assay 92.0 92.0 91.7 91.889.2 89.7 (anhydrous, free base, % w/w) Component Distribution (HPLC %AUC) A₀ + A₁ 2.5 2.5 2.5 2.4 2.4 2.3 B₀ 87.1 87.1 85.6 85.3 84.6 84.3B₁ + B₂ 6.2 5.6 6.2 6.1 6.4 5.5 Related Substances (HPLC % AUC) MAG 0.81.5 1.9 2.1 2.6 4.1 Maximum Individual 0.2 0.2 0.2 0.2 0.2 0.3Unspecified Total Impurities 4.3 4.9 5.8 6.3 6.6 7.9 Moisture (% w/w)18.2 17.4 17.2 16.4 15.4 14.2 pH 2.4 2.5 2.4 2.5 2.5 2.5 MicrobialLimits* 2, 1 cfu/g — — — <1, <1 cfu/g —— Not tested at this time point, per stability protocol.*Total aerobic count, total combined molds and yeast count

TABLE 11A Stability Data for Unformulated Dalbavancin Batch 025 at 25°C./ 60% RH TIME (MONTHS) TEST 0 1 3 6 12 Physical Appearance — — — — —HPLC Assay 92.0 90.9 87.0 85.2 78.8 (anhydrous, free base, % w/w)Component Distribution (HPLC % AUC) A₀ + A₁ 4.0 3.9 3.8 3.7 3.4 B₀ 83.481.6 79.5 76.3 74.9 B₁ + B₂ 7.5 7.4 7.0 7.3 6.3 Related Substances (HPLC% AUC) MAG 1.5 3.6 6.0 8.1 11.7 Maximum Individual 0.2 0.2 0.2 0.4 0.3Unspecified Total Impurities 5.1 7.1 9.6 12.7 15.4 Moisture (% w/w) 11.011.3 12.1 12.9 12.6 pH 3.0 3.0 3.0 3.0 3.0 Microbial Limits* <10, — — —1, <1 cfu/g <10 cfu/g— Not tested at this time point, per stability protocol.*Total aerobic count, total combined molds and yeast count

TABLE 11B Stability Data for Unformulated Dalbavancin Batch 020005/R at25° C./60% RH. TIME (MONTHS) TEST 0 1 3 6 Physical Appearance WhiteWhite White White powder powder powder powder HPLC Assay (anhydrous,free 92.0 91.2 87.2 83.3 base, % w/w) Component Distribution (HPLC %AUC) A₀ + A₁ 2.5 2.5 2.3 2.3 B₀ 87.1 84.9 82.3 78.7 B₁ + B₂ 6.2 5.3 5.35.8 Related Substances (HPLC % AUC) MAG 0.8 3.6 6.1 8.2 MaximumIndividual Unspecified 0.2 0.2 0.3 0.7 Total Impurities 4.3 7.3 10.113.2 Moisture (% w/w) 18.2 15.2 13.8 12.6 pH 2.4 2.6 2.5 2.5 MicrobialLimits* 2.1 — — — cfu/g— Not tested at this time point, per stability protocol.*Total aerobic count, total combined molds and yeast count

Stability tests of the sterilized product were also conducted. Sampleswere tested for appearance of cake and solution, reconstitution time,pH, HPLC assay, bioassay, water content, sterility, HPLC distribution,and related substances. Results are shown in Tables 12-14.

The lyophilized product shows good stability when stored at 5° C. withonly minor changes in HPLC assay or HPLC distribution. (see Tables 12Aand B). As temperature increases (See Tables 13 and 14), there is adecrease in Factor B₀ with a corresponding increase in MAG. The otherfactors do not appear to be affected by temperature. The productappearance and pH do not appear to be affected by temperature. It isalso not possible to determine a clear trend in reconstitution time,considering that the visual inspection of the solubilized product canrequire some seconds. TABLE 12A Stability of Dalbavancin for InjectionLot 149570 at 5° C. (lyophilized, mannitol, pH 3.5) TIME (MONTHS) TEST 03 6 9 12 18 24 36 Physical Appearance White to White to White to Whiteto White to White to White to White to off white off white off white offwhite off white off white off white off white to pale to pale to pale topale to pale to pale to pale to pale yellow yellow yellow yellow yellowyellow yellow yellow solid solid solid solid solid solid solid solidHPLC Assay 97.3 99.8 100.7 100.2 100.0 100.2 99.3 104.4 ComponentDistribution (A₀ + A₁) 3.6 3.6 3.6 3.7 3.6 3.6 3.7 3.7 B₀ 79.4 79.6 80.380.1 80.0 80.1 80.0 81.3 Post B₀* 12.8 12.3 11.3 11.6 11.6 11.7 11.610.6 Degradants MAG 0.6 0.6 0.7 0.6 0.7 0.7 0.7 0.7 Reconstitution Time90 45 40 45 40 40 41 66 Appearance of Clear to Clear to Clear to Clearto Clear to Clear to Clear to Clear to Solution pale pale pale pale palepale pale pale yellow yellow yellow yellow yellow yellow yellow yellowsolution solution solution solution solution solution solution solutionParticulate Matter Particles ≧ — — — — — — — — 10 microns Particles ≧ —— — — — — — — 25 microns Moisture 0.8 1.4 1.2 nd 1.1 1.6 1.5 1.3 pH ofReconstituted 3.7 3.5 3.5 3.5 3.5 3.4 3.4 3.5 Solution Sterility Sterile— — — Sterile — Sterile Sterile— Not tested at this time point, per stability protocol.*Analysis performed with development HPLC method. Dalbavancin B₁ and B₂were not resolved with this method.

TABLE 12B Stability of Dalbavancin for Injection Lot 442378 at 5° C.(lyophilized, mannitol, pH 3.5) TIME (MONTHS) TEST 0 3 6 9 12 18Physical Appearance White to White to White to White to White to Whiteto off off off off off off white to white to white to white to white towhite to pale pale pale pale pale pale yellow yellow yellow yellowyellow yellow solid solid solid solid solid solid HPLC Assay 101.8 102.9100.8 99.9 101.4 99.4 Component Distribution (A₀ + A₁) 2.5 2.5 2.5 2.52.5 2.5 B₀ 85.7 85.5 85.6 85.3 85.3 85.5 (B₁ + B₂) 5.5 5.8 5.6 5.7 6.25.7 Degradants MAG 1.7 1.8 1.6 1.7 1.7 1.9 Maximum Individual n.d. n.d.0.1 n.d. n.d. n.d. Unspecified Total Degradants 1.7 1.8 1.8 1.7 1.7 1.9Reconstitution Time 75 46 43 43 32 32 Appearance Solution Clear to Clearto Clear to Clear to Clear to Clear to pale pale pale pale pale paleyellow yellow yellow yellow yellow yellow solution solution solutionsolution solution solution Particulate Matter Particles ≧ 10 micronsMeets — — — Meets — USP USP Particles ≧ 25 microns Meets — — — Meets —USP USP Moisture 0.3 0.3 0.4 0.7 0.4 0.6 pH of Reconstituted 3.4 3.4 3.43.2 3.4 3.3 Solution Sterility Sterile — — — Sterile —n.d. Not detected— Not tested at this time point, per stability protocol.

TABLE 13A Stability of Dalbavancin for Injection Lot 149570 at 25°C./60% RH (lyophilized, mannitol, pH 3.5) TIME (MONTHS) TEST 0 1 3 6 912 Physical Appearance White to White to White to White to White toWhite to off white off white off white off white off white off white topale to pale to pale to pale to pale to pale yellow yellow yellow yellowyellow yellow solid solid solid solid solid solid HPLC Assay 97.3 99.498.3 100.3 99.7 98.7 Component Distribution (A₀ + A₁) 3.6 3.7 3.6 3.63.6 3.7 B₀ 79.4 81.0 79.3 80.0 79.7 78.9 Post B₀* 12.8 10.6 12.1 11.110.9 11.6 Degradants MAG 0.6 0.8 1.1 1.3 1.8 1.8 Reconstitution Time 9050 45 45 30 40 Appearance of Solution Clear to Clear to Clear to Clearto Clear to Clear to pale pale pale pale pale pale yellow yellow yellowyellow yellow yellow solution solution solution solution solutionsolution Particulate Matter Particles ≧ 10 microns — — — — — — Particles≧ 25 microns — — — — — — Moisture 0.8 nd 1.6 1.4 nd 1.6 pH ofReconstituted 3.7 3.6 3.6 3.5 3.5 3.5 Solution Sterility Sterile — — — —Sterile— Not tested at this time point, per stability protocol.*Analysis performed with development HPLC method. Dalbavancin B₁ and B₂were not resolved with this method.

TABLE 13B Stability of Dalbavancin for Injection Lot 442378 at 25°C./60% RH (lyophilized, mannitol, pH 3.5) TIME (MONTHS) TEST 0 3 6 9 1218 Physical White White White White White White Appearance to off to offto off to off to off to off white white white white white white to paleto pale to pale to pale to pale to pale yellow yellow yellow yellowyellow yellow solid solid solid solid solid solid HPLC Assay 101.8 103.9100.2 98.6 100.7 98.0 Component Distribution (A₀ + A₁) 2.5 2.5 2.4 2.52.4 2.5 B₀ 85.7 85.0 85.5 84.4 84.3 83.9 (B₁ + B₂) 5.5 5.8 5.4 5.7 6.15.6 Degradants MAG 1.7 2.3 2.4 2.8 2.9 3.5 Maximum n.d. n.d. 0.2 n.d.n.d. 0.1 Individual Unspecified Total 1.7 2.3 2.5 2.8 2.9 3.6 DegradantsReconstitution 75 45 38 33 25 31 Time Appearance Clear to Clear to Clearto Clear to Clear to Clear to of Solution pale pale pale pale pale paleyellow yellow yellow yellow yellow yellow solution solution solutionsolution solution solution Particulate Matter Particles ≧ Meets — — —Meets — 10 microns USP USP Particles ≧ Meets — — — Meets — 25 micronsUSP USP Moisture 0.3 0.7 0.6 0.7 0.7 0.8 pH of 3.4 3.4 3.4 3.3 3.4 3.4Reconstituted Solution Sterility Sterile — — — Sterile —n.d. Not detected— Not tested at this time point, per stability protocol.

TABLE 13C Stability of Dalbavancin for Injection Lot 442378 at 30°C./65% RH (lyophilized, mannitol, pH 3.5) TIME (MONTHS) TEST 0 1 3 6 912 Physical White White White White White White Appearance to off to offto off to off to off to off white white white white white white to paleto pale to pale to pale to pale to pale yellow yellow yellow yellowyellow yellow solid solid solid solid solid solid HPLC Assay 100.0 98.599.7 100.0 95.3 96.8 Component Distribution (A₀ + A₁) 2.5 2.4 2.4 2.42.4 2.4 B₀ 86.3 85.6 84.7 84.3 83.2 83.0 (B₁ + B₂) 5.5 5.5 5.7 5.9 6.35.6 Degradants MAG 1.7 2.1 2.5 3.0 3.5 4.2 Maximum 0.1 0.1 0.2 0.2 0.20.2 Individual Unspecified Total 1.8 2.3 2.9 3.3 3.8 4.5 DegradantsReconstitution 45 55 31 27 46 26 Time Appearance Clear to Clear to Clearto Clear to Clear to Clear to of Solution pale pale pale pale pale paleyellow yellow yellow yellow yellow yellow solution solution solutionsolution solution solution Particulate Matter Particles ≧ Meets — — — —— 10 microns USP Particles ≧ Meets — — — — — 25 microns USP Moisture 0.50.7 0.9 0.7 0.9 0.9 pH of 3.5 3.6 3.3 3.4 3.3 3.3 Reconstituted SolutionSterility Sterile — — — — —— Not tested at this time point, per stability protocol.

TABLE 14A Stability of Dalbavancin for Injection Lot 149570 at 40°C./75% RH (lyophilized, mannitol, pH 3.5) TIME (MONTHS) TEST 0 1 3 6Physical Appearance White to White to White to White to off white to offwhite to off white to off white to pale yellow pale yellow pale yellowpale yellow solid solid solid solid HPLC Assay 97.3 98.6 94.7 95.1Component Distribution (A₀ + A₁) 3.6 3.7 3.5 3.5 B₀ 79.4 79.8 77.1 76.5Post B₀* 12.8 10.5 11.9 10.7 Degradants MAG 0.6 2.0 3.7 5.5Reconstitution Time 90 55 45 40 Appearance of Solution Clear to Clear toClear to Clear to pale yellow pale yellow pale yellow pale yellowsolution solution solution solution Particulate Matter Particles ≧10microns — — — — Particles ≧25 microns — — — — Moisture 0.8 n.d. 2.0 1.7pH of Reconstituted Solution 3.7 3.5 3.6 3.5 Sterility Sterile — — —— Not tested at this time point, per stability protocol.*Analysis performed with development HPLC method. Dalbavancin B₁ and B₂were not resolved with this method.

TABLE 14B Stability of Dalbavancin for Injection Lot 442378 at 40°C./75% RH (lyophilized, mannitol, pH 3.5) TIME (MONTHS) TEST 0 1 3 6Physical Appearance White to White to White to White to off white to offwhite to off white to off white to pale yellow pale yellow pale yellowpale yellow solid solid solid solid HPLC Assay 101.8 101.5 98.6 91.8Component Distribution (A₀ + A₁) 2.5 2.4 2.4 2.2 B₀ 85.7 84.2 82.0 80.6(B₁ + B₂) 5.5 5.8 5.6 5.2 Degradants MAG 1.7 3.2 5.3 6.6 MaximumIndividual Unspecified n.d. 0.3 0.2 0.5 Total Degradants 1.7 3.8 5.6 7.4Reconstitution Time 75 46 46 41 Appearance of Solution Clear to Clear toClear to Clear to pale yellow pale yellow pale yellow pale yellowsolution solution solution solution Particulate Matter Particles ≧10microns Meets — — — USP Particles ≧25 microns Meets — — — USP Moisture0.3 0.6 0.8 0.8 pH of Reconstituted Solution 3.4 3.4 3.4 3.4 SterilitySterile — — —n.d. Not detected— Not tested at this time point, per stability protocol.Photostability

Dalbavancin for injection (500 mg vials) were also evaluated forphotostability in labeled vials. Ten labeled vials were introduced intoa photostability chamber, along with a dark control consisting of tenlabeled vials wrapped with aluminum foil. The test samples were exposedto not less than 1.2 million lux hours and an integrated nearultraviolet energy of not less that 200 watt hours/meter². Samples wereevaluated for appearance, appearance of solution, pH, moisture, and HPLCassay. Results of this evaluation are presented in Table 15. TABLE 15Immediate Pack Photostability Results for 500 mg/vial Drug ProductResults Test Light Exposed Dark Control Appearance White to off-whiteWhite to off-white solid solid Appearance of Solution Pale yellowsolution Pale yellow solution Assay (anhydrous, free base) 91.8% 92.8%Moisture Content 0.3% 0.3% pH 4.5 4.5 Reconstitution Time (sec) 43 68Component Distribution (% AUC) A₀ 0.7% 0.7% A₁ 1.7% 1.8% B₀ 86.0% 86.2%B₁ 4.2% 4.3% B₂ 1.9% 2.0% Related Substances (% AUC) C₀ 0.3% 0.3% C₁0.1% 0.1% D₀ 0.2% 0.2% D₁ 0.3% 0.3% IsoB₀ 0.9% 1.0% MAG 1.2% 1.1%Trichloro 0.5% 0.5% RRT 0.85 0.1% n.d. Total Impurities (% AUC) 5.4%5.0%n.d. not detected

Comparison of the light exposed and dark control samples shows that thelight exposed samples remain comparable, with slight changes in potency,API component levels, and total impurities.

The light exposed samples demonstrated the presence of a very low levelof an unspecified impurity at RRT 0.85, which was not detected in thedark control samples. Evaluation of this component by HPLC/MS hasindicated that it is the mono-chlorinated derivative of the dalbavancinB homologues.

Based on these results, Dalbavancin for Injection is consideredphotostable in its immediate pack.

Improved Efficacy and Reduced Side Effects

Weekly dosing of dalbavancin at high dosage levels (i.e., resulting insurprising high and long-lasting serum levels) shows a surprisingly goodsafety profile, similar to, or better than, that observed with thestandard therapy of lower doses of conventional antibiotics administereddaily or even 2-4 times daily, as demonstrated by the Examples herein. Asurprisingly high dosage (i.e., resulting in surprising high andlong-lasting serum levels) of dalbavancin may be administered, with lessfrequency than other antibiotics, and without adverse side effects,enabling improved efficacy and patient compliance.

As discussed in Example 1, treatment with dalbavancin results in a lowincidence of adverse events. Serious adverse events include any adversedrug experience occurring at any dose that results in death, islife-threatening, results in hospitalization or prolongation of existinghospitalization, or persistent or significant disability or incapacity.In the Phase II trial described in Example 1, 90% of adverse reactions,such as diarrhea, nausea, hyperglycemia, limb pain, vomiting, andconstipation, were mild to moderate in severity. Use of dalbavancin inthe trial in Example 1 resulted in no serious adverse events related tostudy drug treatment.

Kits

The invention also provides kits for use in methods of treatment orprophylaxis of bacterial infections. The kits include a pharmaceuticalcomposition of the invention, for example including at least one unitdose of dalbavancin, and instructions providing information to a healthcare provider regarding usage for treating or preventing a bacterialinfection. Instructions may be provided in printed form or in the formof an electronic medium such as a floppy disc, CD, or DVD, or in theform of a website address where such instructions may be obtained.Often, a unit dose of dalbavancin includes a dosage such that whenadministered to an individual, a therapeutically or prophylacticallyeffective plasma level of dalbavancin is maintained in the individualfor at least 5 days. In some embodiments, a kit includes two unitdosages to be administered at least 5 days apart, often about one weekapart, often including a first dosage of dalbavancin that is about 1.5to about 3 times higher than the second dosage. Dalbavancin is oftenincluded as a sterile aqueous pharmaceutical composition or dry powder(e.g., lyophilized) composition.

Suitable packaging is provided. As used herein, “packaging” refers to asolid matrix or material customarily used in a system and capable ofholding within fixed limits a dalbavancin composition suitable foradministration to an individual. Such materials include glass andplastic (e.g., polyethylene, polypropylene, and polycarbonate) bottles,vials, paper, plastic, and plastic-foil laminated envelopes and thelike. If e-beam sterilization techniques are employed, the packagingshould have sufficiently low density to permit sterilization of thecontents.

Kits may also optionally include equipment for administration ofdalbavancin, such as, for example, syringes or equipment for intravenousadministration, and/or a sterile solution, e.g., a diluent such as 5%dextrose, for preparing a dry powder (e.g, lyophilized) composition foradministration.

Kits of the invention may include, in addition to dalbavancin, anon-dalbavancin antibiotic or mixture of non-dalbavancin antibiotics,for use with dalbavancin as described in the methods above.

In the examples below, the following abbreviations have the followingmeanings. If an abbreviation is not defined, it has its generallyaccepted meaning.

-   -   AcOH=acetic acid    -   AcONa=sodium acetate    -   aq.=aqueous    -   AST=aspartate amino transferase    -   ALT=alanine amino transferase    -   BV=bed volume    -   CV=coefficient of variation    -   d=diameter    -   D=dalton    -   DCC=dicyclohexylcarbodiammide    -   DMEPA=3-(dimethylamino)-propylamine    -   DMSO=dimethyl sulfonamide    -   eq=equivalents    -   EU=endotoxin units    -   g=gram    -   GC=gas chromatography    -   HCl=hydrochloric acid    -   H₂O=water    -   HOBT=1-hydroxybenzothiazole hydrate    -   HPLC=high performance liquid chromatography    -   H₂SO₄=sulfuric acid    -   IPA=isopropylamine    -   IU=international unit    -   KF=potassium fluoride    -   Kg=kilogram    -   L=liter    -   LC/MS/MS=liquid chromatography/mass spec/mass spec    -   LDH=lactate dehydrogenase    -   LSC=liquid scintillation counting    -   m³=cubic meter    -   MeOH=methanol    -   mg=milligram    -   mL=milliliter    -   mol=molar    -   MW=molecular weight    -   N=normal    -   NaOH=sodium hydroxide    -   NMP=N-methyl-2-pyrrolidone    -   QTD=quantitative tissue distribution    -   Rt=retention time    -   sd=standard deviation    -   TEA=triethylamine

The following examples are intended to illustrate but not limit theinvention.

EXAMPLES Example 1 Efficacy and Safety of Once Weekly Dalbavancin InDeep Skin and Soft Tissue Infections

This randomized, controlled study evaluated the safety and efficacy oftwo dose regimens of dalbavancin. Adult patients with skin and softtissue infections (SSTI) involving deep skin structures or requiringsurgical intervention were randomized to three groups: Study arm 1received 1100 mg of dalbavancin via intravenous injection (IV) on day 1;Study arm 2 received 1 g of dalbavancin IV on day 1 and 500 mg ofdalbavancin IV on day 8; Study arm 3 received “standard of care.”Clinical and microbiological response and adverse events were assessed.

Populations for Analysis

There were 62 patients randomized into the study; all received at leastone dose of study medication. Four study populations were evaluated forsafety and efficacy and were defined as follows: The intent-to-treat(ITT) population included all patients who received at least one dose ofstudy drug (all randomized study subjects). The microbiologicalintent-to-treat (MITT) population were all ITT patients who had aculture-confirmed Gram-positive pathogen at baseline. Theclinically-evaluable population were defined as those who 1) fulfilledall study entry criteria, 2) had no change in antimicrobial therapy forGram-positive infection following Day 4, except for oral step-downtherapy (only applied to standard of care group), 3) returned for thefollow-up (FU) assessment visit (unless a treatment failure), and 4) didnot receive a non-protocol approved concomitant antimicrobial (unless atreatment failure). The microbiologically-evaluable population was thesubset of clinically-evaluable patients who had a culture-confirmedGram-positive pathogen at baseline.

The study populations are shown in Table 16. TABLE 16 Study Populationsfor Dalbavancin SSTI Treatment Study arm 2 Study arm 1 DalbavancinDalbavancin 1000 mg day 1, Study arm 3 Populations 1100 mg day 1 500 mgday 8 “Standard of care” Randomized 20 21 21 ITT Treated   20 (100%)  21 (100%)   21 (100%) Completed 18/20 (90%) 20/21 (95.2%) 21/21 (100%)Study Clinically eval 16/20 (80%) 17/21 (81%) 21/21 (100%) at EOTClinically eval 13/20 (65%) 17/21 (81%) 21/21 (100%) at FU MITT 14/20(70%) 13/21 (61.9%) 14/21 (66.7%) Micro eval 13/20 (65%) 11/21 (52.4%)14/21 (66.7%) at EOT Micro eval 11/20 (55%) 11/21 (52.4%) 14/21 (66.7%)at FUITT—intent-to-treatMITT—subset of ITT population with culture confirmed Gram-positiveinfectionEOT—end of treatmentFU—follow up

The median age of the subjects was 50-55 years (range 18-86 years).There were no apparent differences in age across the treatment arms.There were differences in gender across treatment arms, but overall thestudy enrolled equal numbers of men and women. The patient populationwas predominantly Caucasian. These results were consistent for both theITT and clinically evaluable populations.

62 patients were enrolled, 20 in Study arm 1 and 21 each in Study arms 2and 3. The most common comparators for standard of care wereclindamycin, ceftriaxone, vancomycin and cefazolin. Mean duration oftreatment in Study arm 3 was 15 days.

Baseline Pathogens and Susceptibility

Of the 62 ITT patients, 66% (14 single-dose dalbavancin, 13 two-dosedalbavancin, 14 standard of care) had a pretherapy Gram-positivepathogen isolated (MITT population). The most common pathogen was S.aureus. The distribution of pathogens at baseline is shown in Table 17.TABLE 17 Baseline Gram-positive Pathogens and Dalbavancin MIC Range forthe MITT Population Single dose Two-dose Number with DalbavancinDalbavancin Standard of Care Pathogen (1100 mg) (1000/500 mg) Regimens(MIC) (N = 14) (N = 13) (N = 14) All S. aureus 13  11  10  (0.12) (0.12)  (0.016-0.25) Methicillin- 7 6 8 sensitive Methicillin- 6 5 2resistant Group B 0 2 2 streptococcus (0.016) (0.016) Streptococcus 0 11 pyogenes (0.016) (0.016) Miscellaneous 3 2 4 Streptococcus (0.016)(0.016) (0.016) and nontypeable strainsClinical and Microbiological Responses

The effectiveness of the three treatment regimens was determined byassessing the patients' clinical response and the documented or presumedmicrobiological responses. The primary efficacy endpoint was clinicalresponse at the follow-up visit for the clinically evaluable population.Clinical response, for both EOT and FU visits, was categorized assuccess (cure or improvement) or failure (including indeterminateresults). Patients classified as successes must not have receivedadditional systemic antibacterial treatment for their infection. Failurewas defined as persistence of one or more local or systemic signs andsymptoms of SSTI such that treatment with new or additional systemicantibacterial agents was required for the SSTI.

Microbiological outcome, a secondary efficacy variable, was assessed inthe subpopulation of patients who had microbiologically documented SSTI(i.e., at least one identified baseline pathogen). A microbiologicresponse was assessed for each Gram-positive pathogen identified atbaseline (i.e., eradication, presumed eradication, persistence, presumedpersistence). For patients for whom follow-up cultures were notperformed, the microbiologic responses for baseline pathogens werepresumed on the basis of the clinical response. Microbiologic responseby patient at the EOT and FU visits was graded as success (i.e., allGram-positive organisms eradicated or presumed eradicated) or failure(i.e., at least one Gram-positive organism persisted or presumed to havepersisted, multiple pathogens with partial eradication). At both the EOTand FU visits, colonization and superinfection were assessed. At the FUvisit, a patient's bacteriological response could also includerecurrence.

Clinical Efficacy

Clinical success rates are shown in Table 18. In theclinically-evaluable population, 61.5% of patients in the single-dosedalbavancin, 94.1% in the two-dose dalbavancin, and 76.2% in thestandard of care group were classified as successes at the time of theFU assessment. In an exploratory subanalysis of those patientscategorized with deep or complicated SSTI at baseline, two-dosedalbavancin therapy also provided a higher clinical success rate(93.8%), compared with the single-dose dalbavancin and standard of caretherapies, 58.3% and 73.7%, respectively.

Similar success rates at both the EOT and FU assessments were found inthe supportive ITT and microbiologically-evaluable populations with aconsistent trend towards a more favorable response following treatmentwith two-dose dalbavancin (Table 17). For the MITT population, clinicalsuccess rates at the FU assessment for those with methicillin-resistantS. aureus (MRSA) were 50% ( 3/6) for single-dose dalbavancin, 80% (⅘)for two-dose dalbavancin, and 50% (½) for patients treated with astandard of care regimen. TABLE 18 Clinical Success Rates by AnalysisPopulation and Treatment Group Single-dose Two-dose (1100 mg) (1000/500mg) Standard of Care Population Dalbavancin Dalbavancin Regimens ITT atEOT 15/20 (75.0) 19/21 (90.5) 17/21 (81.0) ITT at FU 12/20 (60.0) 19/21(90.5) 16/21 (76.2) MITT at EOT 10/14 (71.4) 12/13 (92.3) 10/14 (71.4)MITT at FU  7/14 (50.0) 12/13 (92.3)  9/14 (64.3) Clinically evaluableat 13/16 (61.5) 16/17 (94.1) 17/21 (81.0) EOT Clinically evaluable at 8/13 (61.5) 16/17 (94.1) 16/21 (76.2) FU Microbiologically 10/13 (76.9)10/11 (90.9) 10/14 (71.4) evaluable at EOT Microbiologically  6/11(54.5) 10/11 (90.9)  9/14 (64.3) evaluable at FUMicrobiologic Efficacy

The success rates of the different treatment regimes with respect todifferent pathogens is shown in Table 19. For themicrobiologically-evaluable population, eradication/presumed eradicationrates at the FU assessment for all organisms were 58.3% ( 7/12) forsingle-dose dalbavancin, 92.3% ( 12/13) for two-dose dalbavancin, and70.6% ( 12/17) for patients in the standard of care group. For isolatesthat persisted, there was no change in dalbavancin MIC. At FU, S. aureuseradication rates were higher for the two-dose dalbavancin group (90%)compared with single-dose dalbavancin (50%) and standard of care (60%)treatments. Similar findings were observed for the MITT population;two-dose dalbavancin eradicated 80% of MRSA isolates (Table 19). TABLE19 Success Rates by Pathogen for Microbiologically ITT Population at FUAssessment Single-dose Two-dose (1100 mg) (1000/500 mg) Standard ofDalbavancin Dalbavancin Care Regimens Total organisms 7/16 (43.8%) 14/16(87.5%) 12/17 (70.6%) All S. aureus 5/13 (38%)  9/11 (82%)  6/10 (60%)Methicillin-  2/7 (29%)  5/6 (83%)  5/8 (63%) sensitiveMethicillin-resistant  3/6 (50%)  4/5 (80%)  1/2 (50%) Miscellaneous 2/3 (67%)  4/4 (100%)  5/7 (71%) streptococcus species

For the microbiologically-evaluable and MITT populations, themicrobiological success rates at EOT and FU are summarized in Table 20.Comparable microbiologic success rates were reported at both visits forpatients treated with two-dose dalbavancin and standard of care regimens(approximately 64% to 77%), whereas those given a single dose ofdalbavancin had lower rates of success (<40%). The microbiologic successrates at EOT/FU in the microbiologically-evaluable population paralleledclinical response findings: 38.5%/27.3% for single-dose dalbavancin,72.7%/72.7% for two-dose dalbavancin, and 71.4%/64.3% for standard ofcare therapy. Similar findings were observed for the MITT population(data not shown). TABLE 20 Microbiologic Success Rates Single-doseTwo-dose (1100 mg) (1000/500 mg) Standard of Care DalbavancinDalbavancin Regimens MITT population EOT 5/14 (35.7) 10/13 (76.9)  10/14(71.4) FU 3/14 (21.4) 9/13 (69.2)  9/14 (64.3) Microbiologicallyevaluable population EOT 5/13 (38.5) 8/11 (72.7) 10/14 (71.4) FU 3/11(27.3) 8/11 (72.7)  9/14 (64.3)Pharmocokinetic Analysis

For patients randomized to the dalbavancin treatment groups, 5 ml ofblood was obtained on Day 8 for determination of dalbavancin plasmaconcentrations. For patients randomized to receive a 500 mg dalbavancindose on Day 8, blood was obtained just prior to administration of thesecond dose. Additional 5 ml blood samples were obtained on Days 10 and24 for patients were randomized to the single-dose dalbavancin group andon Days 20 and 34 for those who received two doses of dalbavancin.

Dalbavancin plasma concentrations were determined using validated liquidchromatography and mass spectrophotometer methods. The lower limit ofquantitation was 500 ng/ml for plasma.

Mean dalbavancin concentrations collected on Study days 8, 10, and 24 inthe single-dose regimen were 31.1±7.1, 25.2±4.8, and 10.2±3.5 mg/l(mean±SD), respectively. Dalbavancin concentrations following thetwo-dose regimen on Study days 8 (prior to the second dose), 20, and 34were 30.4±8.2, 21.2±10.0, and 9.0±4.4 mg/l, respectively. As expected,all patients had dalbavancin concentrations of greater than 20 mg/lthrough the first week following the first dose, and levels above 20mg/l were maintained for an additional week with an additional dose of500 mg IV on day 8. Generally, minimum bactericidal concentrations areabout 4 to 10 mg/l.

Safety Evaluation

Each patient who received at least one dose of study drug (ITTpopulation) was evaluated for drug safety through monitoring of adverseevents (AE), including abnormal clinical laboratory test results andvital signs. AE were rated by the investigator as to their severity(mild, moderate, severe, life-threatening), and by the relationship tothe study drug (not related, unlikely related, possibly related, orprobably related).

A summary of the AE data is presented in Table 21. The majority ofadverse reactions (90%) were considered mild to moderate in severity.All serious adverse reactions (8 events in 5 patients) were unrelated tostudy drug treatment. Approximately 59% of all patients who reported atleast one treatment-emergent AE (19 single-does dalbavancin, 16 two-dosedalbavancin, 21 standard of care) experienced an event that wascategorized by the investigator as possibly or probably related to studydrug. Specifically, drug-related AEs were reported in 11 (55%)single-dose dalbavancin, 10 (48%) two-dalbavancin, and 12 (57%) standardof care patients. The most frequently reported drug-related AE in boththe dalbavancin and standard of care treatment groups were diarrhea andnausea. A summary of types of AEs observed for the different treatmentgroups is presented in Table 22.

No dalbavancin-treated patient discontinued treatment prematurely due toan AE. Three of 21 (14%) patients on a standard of care regimentdiscontinued treatment prematurely due to an AE, including one patientwho developed urticaria on Day 1 which was probably drug related and twopatients who had AE unrelated to study drug (superinfection with P.aeruginosa and elevated vancomycin trough level). TABLE 21 Summary ofAdverse Event (AE) Data Single-dose Two-dose dalbavancin dalbavancinStandard of care (N = 20) (N = 21) (N = 21) ≧1 AE 95% 76.2%  100% % AEsevere 15%  9.5%  4.8% ≧1 AE 55%   48%   57% possibly/probably relatedto treatment AE leading to 0 0 14.3% discontinuation of study medication≧1 severe AE 2 (10%) 2 (9.5%) 1 (4.8%)

TABLE 22 Most Common Adverse Events Single-dose Two-dose dalbavancindalbavancin Standard of care (N = 20) (N = 21) (N = 21) Diarrhea 20%9.5% 28.6% Nausea 10% 28.6%  9.5% Hyperglycemia  5% 14.3%   19% LimbPain 10% 9.5%  9.5% Vomiting 10% 14.3%  4.8% Constipation  5% 4.8% 14.3%Discussion

This open-label, randomized Phase II trial shows that dalbavancin iseffective for the treatment of adults with SSTI. The majority ofenrolled patients had deep or complicated infections (>90%) andinfections that required surgical intervention (˜70%), whileapproximately 45% had underlying diabetes mellitus.

Two weekly doses of dalbavancin had a numerically higher clinicalresponse rate than either a single-dose of dalbavancin or the standardof care regimen. Data from both ITT and clinically-evaluable populationssuggests that a regimen of two sequential weekly injectable doses ofdalbavancin (1000 mg, 500 mg weekly) is effective in the treatment ofSSTIs. The standard of care group was treated for a median duration of13 days. At follow-up, 94% of clinically-evaluable patients treated withtwo-dose dalbavancin were considered clinical successes versus 76% ofthose given a standard of care regimen and 61.5% of patients receivingsingle-dose dalbavancin.

S. aureus was the most frequently isolated organism at baseline. In thistrial, approximately 83% of patients were infected with S. aureus and38% of all S. aureus strains were MRSA. Most infections (80%) werecaused by a single pathogen. The MICs for dalbavancin againstGram-positive isolates, including MRSA, ranged from 0.016 to 0.25 mg/L.

Microbiological success rates paralleled those of clinical response forthe clinically-evaluable population. For all organisms combined,treatment with the two-dose weekly dalbavancin regimen provided highereradication rates at the 2 week post-therapy assessment (92%) comparedwith single-dose dalbavancin (58%) and standard of care therapies (71%).Overall, rates of S. aureus eradication were observed in 90%, 50%, and60% of patients, respectively. For the MITT population, rates oferadication for MRSA were 80% for the two-dose dalbavancin regimenversus 50% for both the single-dose dalbavancin and standard of caretherapies.

Concentrations of dalbavancin obtained at the end of the single-dose andtwo-dose weekly treatment periods (Day 10 or Day 20, respectively) weresimilar suggesting little drug accumulation following the second weeklydose. The higher rate of clinical success observed with the two-doseregimen is suggestive of time-dependent killing wherein sustained levelsof drug or drug exposure were provided with two doses of dalbavancinseparated by one week. Dalbavancin plasma levels measured at the end ofthe weekly dosing interval were substantially greater than the reportedMIC₉₀ for pathogens responsible for the majority of SSTIs (<0.03 to 0.5mg/L), including those found in this trial. These levels were also abovethe minimum bactericidal concentrations of 4 to 10 mg/l.

The overall rate of adverse reactions was similar for both dalbavancinregimens and the standard of care group. Gastrointestinal drug-relatedadverse events (i.e., diarrhea and nausea) were most commonly reportedacross the three treatment groups. The majority of these events weremild and self-limited. No dalbavancin-treated patient was withdrawn fromthe study early due to an adverse reaction, nor were any serious adverseevents attributable to the glycopeptide reported, yet 14% of thestandard of care group withdrew due to adverse effects. The novel dosageregimen thus had reduced adverse side effects in comparison to thestandard of care. The data from this trial found no evidence thatdalbavancin induces any degree of clinically significant hepatotoxicityor nephrotoxicity.

The two-dose dalbavancin regimen appears effective for treatment ofpatients with complicated SSTIs. Dalbavancin at both doses was welltolerated in this clinical trial, with an adverse event profile similarto that of the standard of care group.

Example 2 Efficacy and Safety of Once Weekly Dalbavancin in theTreatment of Catheter-Related Blood Stream Infection (CR-BSI)

This study evaluated the safety and efficacy (clinical andmicrobiological) of a weekly dosing regimen of dalbavancin in thetreatment of adults with catheter-related blood stream infection(CR-BSI) due to gram-positive bacterial pathogens, relative to thestandard care of treatment, vancomycin.

Methodology

Patients with CR-BSI due to suspected or known Gram-positive pathogen(s)who met the inclusion/exclusion criteria were randomized to one of twotreatment arms in this open-label, comparative, multi-center study.Dalbavancin was administered once weekly in weekly dalbavancin andcomparator (vancomycin) was administered daily in vancomycin. Catheterremoval was required for patients with Staphylococcus aureus infection;catheter management was left to the Investigator's discretion forpatients with coagulase-negative staphylococci (CoNS) infection.Efficacy was clinically based on the improvement or resolution ofclinical signs and symptoms of CR-BSI such that no additionalantibacterials were warranted, and microbiologically on the eradicationor presence of the baseline or other pathogens. Safety and dalbavancinplasma concentrations were also evaluated.

Population for Analysis

Approximately 80 patients were planned (40 each for treatment arms 1 and3); 67 patients were analyzed (33 in weekly dalbavancin and 34 invancomycin). Seven (7) patients from daily dalbavancin were included insafety analyses only. Seventy-five (75) patients were randomized and 74were treated at 13 sites in the United States; 64 patients completed thestudy. Demographic characteristics were generally similar across studyarms. Mean age for patients in weekly dalbavancin was 54 years (range20-78 years) and in vancomycin was 57 years (range 19-85 years). Malesand females were equally represented overall; there were slightly moremales in weekly dalbavancin and more females in vancomycin. Mostpatients were Caucasian (>65%), were categorized as having probableCR-BSI, and had non-tunneled catheters. For the microbiological ITTpopulation, the most common pathogens for both treatment arms were CoNSand S. aureus. Of the S. aureus isolates, 5/11 (45.4%) and 9/12 (75.0%)were identified as methicillin-resistant (MRSA) for study arms 1 and 3,respectively.

Diagnosis and Main Criteria for Inclusion

Patients with documented Gram-positive bacteremia defined as at leastone blood culture positive for S. aureus, or at least two positive bloodcultures for all other organisms (with at least one of the cultures froma percutaneously drawn sample) were included. In addition, patients werealso enrolled who met all other inclusion criteria, and also met each ofthe two conditions listed below:

-   -   1. At least two of the following signs of bacteremia: core        temperature >38.0° C. or <36.0° C., measured rectally, orally        (added 0.5° C. to the measured temperature), tympanically or via        a central catheter; WBC count >12,000/mm³ or <4,000/mm³ or        differential count showing >10% band forms; tachycardia (pulse        rate >100 bpm); tachypnea (respiratory rate >20.        breaths/minute); transient hypotension (systolic blood pressure        <90 mm Hg)    -   2. No apparent source for the clinical manifestation of        bacteremia other than the catheter (may have local signs and        symptoms at the catheter site).        Treatment

The treatment lasted for 14 days for patients with S. aureus infection,and 7 to 14 days for all other pathogens. Because of the long half-lifeof dalbavancin, the duration of study drug therapy was assumed to be 7days for each dose of dalbavancin given in weekly dalbavancin.Dalbavancin was administered intravenously with a 1000 mg loading doseon Day 1 and a 500 mg dose on Day 8. Vancomycin was administeredintravenously with a 1000 mg dose every 12 hours (or dose-adjusted basedon drug levels).

Criteria for Evaluation

Efficacy was evaluated based on clinical and microbiological responses.The primary outcome parameter was overall response at the test-of-cure(TOC) visit in the microbiological intent-to-treat (ITT) population.Safety was evaluated by the collection and analysis of data on adverseevents (AEs), clinical laboratory tests, physical examinations, vitalsigns, and ECGs. Dalbavancin plasma concentrations were determined on upto seven occasions (prior to and after the first dose on Day 1, on Day4±2 days, before and after the second dose on Day 8, at end of treatment(EOT) and at TOC. Due to the elimination of arm 2 during the study, onlydemographic data and safety data from those patients are described;efficacy was not evaluated.

Statistical Methods

Efficacy, safety, and dalbavancin concentration data are presented usingdescriptive statistics. For the primary efficacy analysis, 95%confidence intervals were also determined, and for prognostic factoranalyses using the primary efficacy variable, logistic regression wasused.

Efficacy Results

For the primary efficacy analysis, overall response in themicrobiological ITT population at TOC, patients who received dalbavancinin weekly dalbavancin (87.0%, 95% CI: 73.2, 100.0) had a higher successrate than patients who received vancomycin (50.0%, 95% CI: 31.5, 68.5).

For the secondary efficacy analyses, overall success, clinical success,and overall and clinical success by pathogen, by category of infection,by catheter status at baseline, and by type of catheter for themicrobiological ITT and evaluable populations at EOT and TOC were higherin the dalbavancin study arm compared with the vancomycin study arm.By-patient microbiological success was similar for the treatment arms atEOT, but was higher for the dalbavancin study arm at TOC. For CoNS,by-pathogen microbiological success was greater in the dalbavancin studyarm at both EOT and TOC. By EOT, most signs and symptoms at the cathetersite were resolved for both study arms.

Safety Results

Adverse events (AEs) were reported by 71 patients (95.9%). The number ofpatients reporting AEs was similar across study arms, although more AEswere reported in the dalbavancin groups than in the vancomycin group.The most common AEs were diarrhea, constipation, anemia, andhypotension. Most AEs were considered by the Investigator to be mild ormoderate in intensity. Adverse events considered to be related (possiblyor probably) to study drug were evenly distributed across study arms.One (1) patient (3%) in the dalbavancin study arm had an unrelated,non-serious AE that led to discontinuation of dalbavancin; no AEs led towithdrawal from the study. Three (3) patients (8.8%) in the vancomycinstudy arm had AEs that led to discontinuation of comparator; 2 AEs ledto withdrawal from the study. The distribution of SAEs was similar amongtreatment groups; no SAE in the dalbavancin study arm and one SAE in thevancomycin study arm was considered to be related to study drug. Therewere 5 deaths in this study. All AEs that resulted in death wereunrelated to study medication. There were no clinically meaningfullaboratory abnormalities in any study arm. Few laboratory values werereported as AEs. None were SAEs or led to discontinuation of study drug.

Increased diastolic blood pressure was the most common abnormalclinically significant change across all treatment groups. Hypertensionwas reported as an AE for 3/74 patients (4.1%, 2 patients ondalbavancin, 1 patient on vancomycin); only 1 patient (receivingdalbavancin) had increased blood pressure as an AE considered related tostudy medication. Dalbavancin did not exhibit any impact on heart rate,atrioventricular conduction or intra-ventricular conduction. The averagedifference of effect on QTc values was 7 msec greater for thedalbavancin group in comparison with the vancomycin group. Nosignificant difference was observed between treatments for frequency ofoutliers during drug therapy.

Therefore, dalbavancin given as an initial IV dose of 1000 mg followed 1week later by a second IV dose of 500 mg appears well tolerated andhighly effective for the treatment of CR-BSI caused by Gram-positivepathogens, with superior response rates to vancomycin

Example 3 Pharmacokinetics and Renal Excretion of Dalbavancin in HealthySubjects

The primary objectives of this study were to characterize thepharmacokinetics of dalbavancin and to calculate the extent of renalexcretion in healthy subjects receiving a therapeutic dose of the drug.This was an open label, non-comparative, study.

Study Drug Treatment

Healthy male or female subjects between 18 and 65 years of age wereadministered a single 1000 mg IV dose of dalbavancin infused over 30minutes.

Six subjects, one female and five male, were enrolled, received studymedication, and completed all aspects of the study. Three subjects wereCaucasian and three subjects were African-American. Mean age was 29.8years (range 22 to 63). Mean height was 68.6 inches (range 63 to 75) andmean weight was 179.6 lbs (140 to 244).

Pharmacokinetics

Blood and urine (24-hr collections) were collected on study days 1, 2,3, 4, 5, 6, 7, 14, 21, 28, and 42. Blood samples were drawn intoheparinized tubes and centrifuged. Plasma was separated and storedfrozen at −20° C. until time of assay. Plasma and urine samples wereassayed for dalbavancin using validated LC/MS/MS methods. The lowerlimit of quantitation of the assay was 500 ng/mL for urine and plasma.

Dalbavancin pharmacokinetic parameters were estimated bynon-compartmental methods using the WinNonlin™ software (PharsightCorporation). The peak concentration (C_(max)) values were obtaineddirectly from the observed data. The area under the plasmaconcentration-time curve (AUC) was calculated using the lineartrapezoidal rule. Clearance (CL) was computed as dose/AUC. Theelimination half life (t_(1/2)) was estimated by linear regression ofthe log-linear portion of the log concentration versus time curve.Estimates of the volume of distribution (Vz) was calculated using theregression parameters, while the volume of distribution at steady state(Vss) was calculated from the area under the first moment curve (AUMC)multiplied by the dose and divided by AUC. The cumulative amount ofdalbavancin excreted in urine was determined as the integrand of theurine excretion rate (AURC). CL_(R), or renal clearance, was calculatedas the ratio: CL_(R)=AURC/AUC.

Plasma concentrations of dalbavancin versus time are shown for allsubjects in FIG. 5. Pharmacokinetic parameters are presented in Table23. Concentrations were similar across all subjects. Peak plasmaconcentrations were approximately 300 mg/L and were achieved immediatelyfollowing the end of infusion. Dalbavancin shows an apparent volume ofdistribution of more than 10 L and is assumed to be well distributed inthe extracellular fluid.

Dalbavancin was slowly eliminated with a t_(1/2) of 9-12 days. The totaldrug clearance was 0.0431±0.0074 L/hr. The estimated fraction of drugexcreted unchanged into urine was 42% of the administered dose, andrenal clearance was estimated as 0.018 L/h. The variability observedacross subjects was low with a coefficient of variation of less than 22%across all pharmacokinetic parameters. TABLE 23 Pharmacokineticparameters Cmax t_(1/2) AUC V_(Z) CL V_(SS) AURC % Renal CL_(R) mg/L hmg · h/L L L/h L mg Excretion L/h Mean 301 257 23843 16.0 0.0431 11.5419 41.9 0.0181 Sd 65 21 4526 3.1 0.0074 2.13 27 2.7 0.0036 CV % 21.68.1 19.0 19.5 17.1 18.6 6.4 6.4 20.1 Min 243 227 19844 11.7 0.0332 8.58379 37.9 0.0130 Max 394 282 30100 19.6 0.0504 13.9 448 44.8 0.0226Safety Assessments

Adverse events were recorded and assessed for severity and relationshipto study drug. Laboratory data (chemistry panel, CBC with differential,urinalysis) were collected and assessed for changes from baseline andout-of-range values. ECG, physical examination, and vital signs wereobtained, and changes from baseline were assessed.

Dalbavancin was well-tolerated in this study. No subject deaths orserious adverse events were reported during this study and no subjectwas prematurely withdrawn from study due to an AE.

All volunteers reported at least one AE, all of mild intensity. Threevolunteers reported AEs that were possibly related to study medication:elevated ALT (value 46 IU/L, upper limit of normal 40 IU/L) in onesubject; eosinophilia (value 0.5×10³ μL, upper limit of normal0.4×10³/μL), elevated LDH (value 303 IU/L, upper limit of normal 90IU/L), elevated ALT (value 54 IU/L, upper limit of normally 40 IU/L),elevated AST (value 42 IU/L, upper limit of normal 40 IU/L) all in onesubject; and tinnitus in one subject.

No trends were seen for post-baseline hematology, chemistry, vitalsigns, and ECG results.

Discussion

A single 1000 mg IV dose of dalbavancin was well-tolerated. Following asingle intravenous infusion of 1000 mg, plasma concentrations ofdalbavancin above 45 mg/l are maintained for at least seven days. Thisis above concentrations known to be bactericidal (4-32 mg/l). Thissupports the use of dalbavancin as a once-weekly regimen. The urinaryelimination profile indicates that renal excretion is an importantelimination pathway, with approximately 40% excreted in urine. Thisfinding is consistent with observations in animals. Since the kidneysare not the exclusive elimination route, a dosing adjustment fordalbavancin may not be necessary in renally impaired patients.

Example 4 Pharmacokinetics of Dalbavancin Subjects with Renal Impairment

These were open label studies conducted to examine the safety andpharmacokinetics of intravenous dalbavancin when administered tosubjects with mild, moderate and severe renal impairment.

Study Drug Treatment

Male and female subjects between ages 18 and 80 were eligible forenrollment. Subjects had to be within −10% to +50% of ideal body weightfor their sex, height, and body frame.

Pharmacokinetics

Serial blood samples were collected before the dose and through at least2 weeks after the end of infusion and assayed for dalbavancin using avalidated LC-MS/MS method.

Dalbavancin pharmacokinetic parameters were estimated bynon-compartmental methods. The peak concentrations (C_(max)) wereobtained directly from the observed data. The area under the plasmaconcentration-time curve (AUC) were calculated using the lineartrapezoidal rule.

Pharmacokinetic parameters of subjects with healthy renal impairment andsubjects with the most severe renal impairment (Severe RI) are presentedin Table 24. TABLE 24 Pharmacokinetic parameters Dosing RegimenPharmacokinetic 1000 mg + 500 mg 1000 mg Parameter 1000 mg 1120 mg 500mg Severe RI{circumflex over ( )} Severe RI* AUC (mg · h/L) 24453 +/−3711 25790 +/− 2447 33000{grave over ( )} 24376 +/− 6615 ˜48000{graveover ( )} AUC7 (mg · h/L) 13412 +/− 2120 13644 +/− 1305 12000{grave over( )}  6860 +/− 1613 ˜14000{grave over ( )} AUC14 (mg · h/L) 17737 +/−2325 20207 +/− 2122 23000{grave over ( )} 10986 +/− 2817 ˜22000{graveover ( )} AUC42 (mg · h/L) 23137 +/− 3326 33000{grave over ( )}˜38000{grave over ( )} Cmax (mg/L) 340 +/− 68 325 +/− 41  300{grave over( )} 137 +/− 22  ˜300{grave over ( )} C7day (mg/L)    40.9 +/− 5.2^(@)55.4 +/− 8.1   40{grave over ( )} ˜30-40   ˜57{grave over ( )} C14day(mg/L)    21.3 +/− 2.5^(@) 22.8 +/− 1.9   40{grave over ( )}   ˜40{graveover ( )}{circumflex over ( )}Preliminary clinical data*Estimated using parameters directly referenced, or extrapolated fromplots{grave over ( )}Based on simulated data or extrapolated based on anotherstudy; not directly referenced in an abstractAUC = drug exposure as estimated by the area under the plasmaconcentration-time curve;AUC7 = drug exposure through 7 days post-dose or through treatmentperiod;AUC14 = drug exposure through 14 days post-dose or through treatmentperiod;AUC42 = drug exposure through 42 days post-dose or through treatmentperiod;C_(max) = maximum observed drug concentration in plasma;C7 = drug concentration 7-days post-dose, prior to administration ofanother possible dose;C14 = drug concentration 14 days post-dose, prior to administration ofanother possible dose.Discussion

For patients with severe renal impairment, a single dose of 500 or 1000mg of dalbavancin is administered to the subject. As apparent from Table24, after 14 days, a patient with severe renal impairment given a singledose of 1000 mg has a AUC14 (mg.h/L) of approximately 22000, which isunexpectedly very similar to normal patients on the two dose regimen of1000 mg+500 mg (AUC14 mg.h/L of 23000).

For patients with mild renal insufficiency, no dosage adjustment ofdalbavancin was required. Dalbavancin concentrations and pharmacokineticparameters were similar in subjects with mild renal impairment andsubjects with normal renal function. In addition, dalbavancin waswell-tolerated in subjects with normal or mildly impaired renalfunction. See Dowell, J. et al. “Dalbavancin Dosage Adjustments NotRequired for Patients with Mild Renal Impairment” presented at the 2003ECCMID Meeting, Glasgow (Clinical Microbiology and Infection, Volume 9(Supplement 1) page 291; 2003) and Stogniew, M. et al. “PharmacokineticAttributes of Dalbavancin: Well Distributed and Completely Eliminatedwith Dual Routes of Elimination” Presented at the 2003 ECCMID Meeting,Glasgow (Clinical Microbiology and Infection, Volume 9 (Supplement 1)page 291-292; 2003) both of which are hereby expressly incorporated byreference in their entirety.

Example 5 Renal Impairment and End-Stage Renal Disease

A major pathway for dalbavancin elimination is the excretion of bothintact and OH-dalbavancin into urine, and it is likely that the drugwill be used in patients with various degrees of renal impairment.Because of this, the safety and pharmacokinetics of dalbavancin insubjects with various degrees of renal impairment was examined inClinical Studies VER001-3, VER001-11 and VER001-13.

Clinical Study VER001-3 was terminated early (5 subjects enrolled; 3subjects received dalbavancin) when it was determined that the dosagebeing examined (70 mg) was too low compared to the larger weekly dosagethat was being proposed for clinical study. Clinical Study VER001-13examined subjects with mild and moderate renal impairment and ClinicalStudy VER001-11 examined subjects with severe renal impairment andsubjects with end-stage renal disease (ESRD). Matched control subjectswere enrolled in all studies. Renal impairment was defined by estimatedcreatinine clearance: mild =51-79 mL/min, moderate=31-50 mL/min, andsevere <30 mL/min. Subjects with ESRD were dependent on dialysis throughthe course of the study (3 times weekly). Single doses of 1000 mg IVdalbavancin were examined in subjects with mild and moderate renalimpairment, doses of 500 mg dalbavancin were studied in subjects withESRD, and single doses of 500 and 1000 mg were studied in subjects withsevere renal impairment.

Dalbavancin was well tolerated in each of these renal impairmentstudies. Similar percentages of subjects reporting adverse events wereobserved in each of the study groups. The majority of the reportedadverse events were mild or moderate in severity and unrelated to studymedication. There were no clinically meaningful laboratory derangementsin any of the study subjects while receiving dalbavancin.

In Clinical Study VER001-13, dalbavancin administration to subjects withnormal renal function and subjects with mild renal impairment resultedin comparable concentration-time profiles over the 60 day samplinginterval. Through the relative treatment period, 7-days postdose,concentration-time profiles were comparable among subjects with normalrenal function, and subjects with either mild or moderate renalimpairment. A small increase in concentrations was observed in subjectswith moderate renal impairment beyond Day 14, when concentrations wererelatively low and below 40 mg/L. Dalbavancin plasma concentration-timeprofiles in these subjects following 1000 mg dalbavancin are shown inFIG. 18. Dalbavancin pharmacokinetic parameters are shown in Table 25.No dosage adjustment is required for patients with mild or moderaterenal impairment (CL_(CR)>30 mL/min). The findings from this study areconsistent with patients studied in the population pharmacokineticanalysis (CL_(CR)>50 mL/min).

Dalbavancin was administered to subjects with normal renal function,subjects with severe renal impairment, and ESRD subjects in ClinicalStudy VER001-11. Dalbavancin concentration-time profiles observed inthis study are shown in FIG. 19. Concentrations were similar amongsubjects with normal renal function and subjects with either severerenal impairment or ESRD through the first 12 hours, but a small andgradually increasing difference was observed in subjects with severerenal impairment. Concentrations were increased by ≦40% through thefirst week post-dose, and differences continued to increase through theremainder of the profile. Subjects with ESRD were divided into subjectsreceiving dalbavancin before or after their first dialysis session. Nodifference in concentration-profiles was noted between these twosubgroups. Concentrations in subjects with ESRD were, however, similarto subjects with normal renal function, indicating compensation in renalinsufficiency due to regularly scheduled dialysis (3-times/week). Levelsin dialysate corresponding to this level of drug dialysis were too smallto measure.

Dalbavancin pharmacokinetic parameters in subjects with severe renalimpairment and subjects with ESRD (VER001-11) are compared to subjectswith mild or moderate renal impairment (VER001-13) in Table 25. Singledoses of 500 and 1000 mg were studied in subjects with severe renalimpairment; concentrations and exposures were consistent with doseproportionality (FIG. 20, Table 25). Individual drug exposures withinthe relative treatment period (AUC_(0-Day7)) were generally consistentacross individual CL_(CR), with only a small increase in exposureobserved for subjects with severe renal impairment (FIG. 21).Differences in exposures were much greater when examined across theentire concentration-time profile. Area under the plasmaconcentration-time curve extrapolated through infinity (AUC_(0-inf)) wasincreased 97% in subjects with severe renal impairment. Subjects withESRD had only a 45% increase in AUC_(0-inf), reflecting somecompensation in renal insufficiency with regularly scheduled dialysis.

Based on the pharmacokinetic parameters and simulations based on thedata, a dosage adjustment is recommended for patients with severe renalimpairment. The intent of the dosage adjustment is to matchconcentrations and exposures during the two-dose relative treatmentperiod (14 days), while minimizing the overall exposure of drug. A totalof 9 simulated dosage regimens were examined in subjects with severerenal impairment. The results of these simulations are summarized inFIG. 22, showing the exposure during the relative treatment periodversus the overall exposure. A dose of 750 mg dalbavancin followed oneweek later by 250 mg dalbavancin is the recommended dosage adjustmentfor patients with a CL_(cr)<30 mL/min. This dosage regimen maintainsconcentrations above 20 mg/L, matches the treatment exposures observedfor subjects with normal renal function, and minimizes the overallexposure. FIG. 23 shows dalbavancin concentration-time profilessimulated for i) subjects with normal renal function receiving 1000 mg(Day 1)+500 mg (Day 8), ii) subjects with severe renal impairmentreceiving 1000 mg (Day 1)+500 mg (Day 8), and iii) subjects with severerenal impairment receiving the recommended dosage adjustment of 750 mg(Day 1)+250 mg (Day 8). Patients receiving regular dialysis therapy (2to 3 times weekly) have some compensated clearance of drug by dialysisand do not require a dosage adjustment. TABLE 25 DalbavancinPharmacokinetic Parameters for Healthy and Renally Impaired SubjectsFOLLOWING A single dose of dalbavancin Moderate Normal Severe SevereMild Renal Renal Renal Renal Renal ESRD ESRD Normal Renal Normal RenalMean Impairment Impairment Function (N = Impairment ImpairmentPredialysis Postdialysis Function Function (± SD) (N = 6) (N = 6) 9) (N= 6) (N = 4) (N = 3) (N = 3) (N = 6) (N = 3) Study VER001- VER001-13VER001-13 VER001- VER001- VER001-11 VER001-11 VER001-11 VER001-2 13 1111 Dose  1000  1000  1000  500  1000  500  500  500  1120 (mg) C_(max) 266.8  330.7  248.8  136.5  315.3  140.7  145.8  137.3  325 (mg/L)(42.3) (55.7) (33.0) (21.6) (89.7) (26.4) (71.5) (39.5) (41)AUC_(0-day 7)  9714 11050  8992  6077 10653  6069  4969  5245 13644 (mg· h/L) (1406) (2005) (1362) (1392) (1474) (1768) (1153) (1661) (1305)AUC_(0-day 14) 15333 18060 13765 10412 18698 10620  8500  7971 20207 (mg· h/L) (1884) (3065) (1986) (2658) (1780) (2881) (2297) (2422) (2122)AUC_(0-inf) 27047 37665 24561 24074 44497 19772 15587 12219 25790 (mg ·h/L) (4084) (7123) (5252) (6613) (11483) (5065) (6050) (3298) (2447) CL  0.0376   0.0273   0.0422   0.0222   0.0238   0.0264   0.0350   0.0429  0.0437 (L/h) (0.0048) (0.0045) (0.0079) (0.0064) (0.0068) (0.0063)(0.0113) (0.0092) (0.004) Vss   16.5   14.7   18.5   13.2   14.2   12.8  14.6   15.0   8.49 (L) (3.3) (2.1) (3.6) (2.9) (0.8) (3.4) (3.2) (4.2)(1.05) t_(1/2)  389  432  417  454  469  376  347  333  149 (h) (59)(43) (108) (102) (103) (63) (78) (91) (3.6)

Example 6 Hepatic Impairment

Dalbavancin is eliminated by both renal and non-renal pathways anddalbavancin will likely be used in patients with various degrees ofhepatic impairment. The safety and pharmacokinetics of dalbavancin insubjects with various degrees of hepatic impairment was examined inClinical Study VER001-12. The study enrolled otherwise healthy subjectswith mild, moderate, or severe hepatic impairment (Child-Pugh scores 5to 6, 7 to 9, and 10 to 15, respectively) and matched control subjectswith normal hepatic function. Subjects were given a single 1000 mg IVdose of dalbavancin on Day 1 followed by a 500 mg IV dose of dalbavancinon Day 8.

Concentrations and exposures of dalbavancin did not increase withincreasing degrees of hepatic impairment (FIG. 24). Dalbavancinadministration to subjects with normal hepatic function and subjectswith mild hepatic impairment resulted in comparable concentration-timeprofiles over the 60 day sampling interval. Dalbavancin administrationto subjects with moderate hepatic impairment and subjects with severehepatic impairment resulted in slightly decreased observedconcentrations when compared to subjects with normal hepatic function.The drug was well tolerated across all groups.

The exposure to dalbavancin for subjects with normal hepatic functionand subjects with mild hepatic impairment was comparable (Table 26). Atrend towards a decrease in dalbavancin exposure and an increase in CLwas evident for subjects with moderate hepatic impairment and subjectswith severe hepatic impairment. Overall, the intersubject variability ofthe pharmacokinetic parameters was low and generally below 30% andalthough dalbavancin pharmacokinetic parameters were statisticallydifferent between the higher and lower hepatic impairment groups, theranges of the exposure parameters significantly overlapped (FIG. 25).

Changes in dalbavancin pharmacokinetic parameters appeared to beinfluenced by the drug's distribution volume. Volumes of distributionincreased in the same proportional or inversely proportional manner asCL and AUC. The drug terminal elimination half-life was virtuallyunchanged across the groups. Subjects with moderate and severe hepaticimpairment, who have significantly more ascites and edema, have largervolumes of drug distribution and subsequently slightly lower drugexposures.

There was overlap in concentrations through the profiles across groups,with mean concentrations remaining above 20 mg/L in all groups throughthe intended treatment duration of 14 days. There was also overlap indrug exposure across the groups (FIG. 25). Even with a decrease in meanexposure, subjects with severe hepatic impairment still had a drugexposure through the relative treatment period (14 days) that exceededparameters required for therapy. No dosage adjustment of dalbavancin isrequired for patients with any degree of hepatic impairment. TABLE 26Dalbavancin Pharmacokinetic Parameters for Healthy and HepaticallyImpaired Subjects FOLLOWING 1000 MG dalbavancin ON Day 1 and 500 MGdalbavancin on Day 8 Mild Hepatic Moderate Hepatic Severe Hepatic NormalHepatic Impairment Impairment Impairment Function Mean (± SD) (N = 6) (N= 6) (N = 5) (N = 9) C_(max1) (mg/L)  331.7 (80.6)  227.2 (37.5)  199.0(30.4)  278.3 (52.6) C_(max2) (mg/L)  177.0 (62.4)  122.7 (27.2)  118.9(23.9)  166.3 (42.9) AUC_(0-day8) (mg · h/L)  11146 (3478)   7710 (1099)  7561 (1540)  10577 (2493) AUC_(0-day15) (mg · h/L)  21158 (5808) 14826 (1925)  14112 (2911)  20473 (4883) AUC_(0-inf) (mg · h/L)  33117(6479)  23628 (3527)  21639 (5940)  33851 (8184) CL (L/h) 0.0466(0.0084) 0.0647 (0.0098) 0.0736 (0.0202) 0.0466 (0.0110) Vss  18.1 (5.2) 24.4 (3.0)  25.2 (4.0)  18.3 (3.7) t_(1/2) (h)   323 (27)   320 (24)  322 (68)   321 (24)

Example 7 Protein Binding of Dalbavancin using Isothermal TitrationMicrocalorimetry

Binding of dalbavancin to proteins was measured by isothermal titrationmicrocalorimetry (ITC) in 20 mM phosphate, 150 mM NaCl, pH 7.4 at 25 and37° C. using a Microcal VP-ITC instrument. In a typical experiment,25×10 μl of protein (˜150 μM) was injected into a calorimeter cellcontaining dalbavancin solution (˜5 μM). Actual protein and dalbavancinconcentrations were determined by measuring absorbence at 280 nm.Control experiments included injections of protein into buffer (in theabsence of dalbavancin) to account for the heats of dilution of proteinunder identical conditions. For comparison, similar experiments withsome necessary modifications were performed using teicoplanin.

Experiments with dalbavancin were conducted with each of the followingproteins: human albumin; dog albumin; rat albumin; bovine albumin; andhuman α-glycoprotein. Teicoplanin was studied with human albumin andα-glycoprotein. A comparison of binding affinities at two differenttemperatures is shown in Table 27. TABLE 27 Comparison of apparentbinding affinities (Ka, ×10⁵ M⁻¹) 25° C. 37° C. Dalbavancin Humanalbumin 1.35 (±0.2) 1.33 (±0.15) Rat albumin  3.1 (±0.5)  2.8 (±1.8) Dogalbumin 0.62 (±0.09) 0.50 (±0.13) Bovine albumin 1.38 (±0.14)α-glycoprotein 1.84 (±0.36)  4.8 (±2.3) Teicoplanin Human albumin 0.96(±0.08) α-glycoprotein 0.07 (±0.01)The ± errors quoted are the standard deviations obtained from thefitting routine.

Integrated heat effects, after correction for heats of dilution, wereanalyzed by non-linear regression using a simple single-site bindingmodel with the standard Microcal ORIGIN software package. Raw data(μcal/sec) for each injection were integrated to give the total heateffect per addition, then divided by amount of injectant to givekcal/mole of injectant. The same integration was applied for controldilution effects, and this was subtracted from the actual titrationdata. This provided a differential of the binding curve in which theextent of binding is proportional to the total heat liberated (orabsorbed). This was then analyzed by non-linear regression methods interms of various standard binding models. The simplest model assumessimple non-competitive binding equilibrium, and gives three parameters:

-   -   K_(a) (=1/Kdiss) is the binding association (dissociation        constant)    -   ΔH=the enthalpy of binding (the size of signal related to        binding)    -   N=number of binding sites (assuming the binding model is        correct)

Assuming non-competitive binding, N is the (relative) number of moles ofinjectant required to saturate all the available binding sites in thesample. For the dalbavancin experiments, dalbavancin is the “sample” andthe protein (HSA, etc.) is the “injectant.” These preliminary resultsindicate that the binding is relatively weak and, because of the poorsolubility of dalbavancin, it is difficult to determine the bindingstoichiometry (N) unambiguously. However, as seen in FIG. 6, in allcases, the data fits well with N<1 (i.e., less than one to one proteinto dalbavancin). Consequently, a value of N=0.5 means that it only takeshalf as many moles of protein to bind all the dalbavancin as would beexpected. In other words, each protein apparently binds two dalbavancinmolecules. It is possible that dalbavancin forms a dimer that binds 1:1with a protein. Results of binding stoichiometry modeling suggests thattwo dalbavancin molecules are bound to one molecule of protein, unliketeicoplanin, which exhibits 1:1 binding.

Table 28 presents the calculated percent bound for antibioticconcentrations in the range 1-500 μM, assuming physiologicalconcentrations of human serum albumin (6×10⁻⁴ M) and α-glycopeptide(1.5×10⁻⁵ M). To relate this to the clinical situation, the peakconcentration of dalbavancin is approximately 300 mg/L, or 165 μM. TABLE28 Calculated percent Binding of Teicoplanin and Dalbavancin to PlasmaProteins Concentration of antibiotic (μM) Dalbavancin Teicoplanin Humanalbumin  1 98.8 98.3   10 98.8 98.3  100 98.7 98.0  165 98.6 ND 250 98.5ND 500 98.0 ND Human α-glycoprotein  1 73.0 9.6  10 68.2 9.1 100 26.26.0 165 16.9 ND 250 11.4 ND 500 5.9 NDND = Not done

In these experiments, the binding of dalbavancin to human serum albuminexceeds 98%. The fraction bound is fairly constant across the selectedrange of dalbavancin concentrations, i.e. 1-500 μM. This rangeencompasses the therapeutic concentrations in man. Binding ofdalbavancin to α-glycoprotein is much greater than that of teicoplanin.Dalbavancin demonstrates high capacity and low affinity for plasmaproteins of different origin, with similar K_(a) values across proteinsfrom all species tested. These results help to explain some of theunique pharmacokinetic characteristics of dalbavancin. The binding andformation of a 2:1 dalbavancin:protein complex also explains theprolonged half-life, and the apparent volume of distribution, whichapproximates extracellular water volume. The low affinity helps explainthe observed in vivo activity, which greatly exceeds what would beexpected for a compound with a free fraction close to 1%. The highcapacity for plasma proteins helps to explain the relatively high plasmaconcentrations achieved in spite of poor solubility of the compound atphysiological pH.

Example 8 Pharmacokinetic Attributes and Tissue Distribution ofDalbavancin in Rats

Two studies were performed in rats administered a single IV infusion of20 mg/kg [³H]-dalbavancin. Excreta and more than 40 different tissueswere collected through 70 days post-administration, and the tissuedistribution and pharmacokinetics of drug-derived radioactivity weredetermined.

HPLC-purified [³H]-dalbavancin was used for these studies. Radiolabeleddrug was produced via tritium exchange and purified by HPLC.

Rat Mass Balance Study

Mass balance studies were conducted to determine the excretion patternof dalbavancin following a single intravenous (IV) infusion ofdalbavancin in male rats.

Fifteen male Sprague-Dawley rats received a single IV dose of³H-dalbavancin (20 mg/kg, 100 μCi/rat). Following dose administration,urine and feces were collected at 24 hour intervals to 14, 36, and 70days after the dose (3 rats/final collection time). Water and methanolcage washes were also collected. Carcasses were analyzed at the end ofthe collection period. All samples were analyzed for total radioactivitycontent by liquid scintillation counting (LSC).

Following IV administration of ³H-dalbavancin in rats, drug-derivedradioactivity was eliminated in both urine (˜⅔ of excretedradioactivity) and feces (˜⅓ of excreted radioactivity). Approximatelyhalf of the radioactivity administered was eliminated in the urine andfeces within the first week, which is consistent with a plasma t_(1/2)of approximately 1 week. At 70 days post dose, only 4.5% of the doseremained in the carcass. Negligible radioactivity was recovered in thecage washes. Virtually all of the administered radioactivity wasaccounted for (urine, feces, carcass, cage washes, and tritium exchange)during the study.

Rat Quantitative Tissue Distribution (OTD) Study

Quantitative tissue distribution studies were conducted to assess thetissue distribution of dalbavancin following a single IV infusion ofdalbavancin to male rats.

Forty-one male Sprague-Dawley rats received a single IV infusion of³H-dalbavancin (20 mg/kg, 50 μCi/rat). Rats (3 per time-point) wereeuthanized at 12, 24, 48, 72, 96, 120, 144, 168, 336, 840, 1176 and 1680hours post dose for collection of blood, plasma, and tissues (includingcarcass). All samples were analyzed by LSC.

Concentration-time profiles were determined for more than 40 tissues,including kidney, liver, spleen, blood, plasma, lung, and skin.Concentrations and t_(1/2) values of drug-derived radioactivity intissues, including skin, were comparable to those observed in plasma.Dalbavancin was found to be rapidly and extensively distributed with alltissues having quantifiable concentrations of drug-derived radioactivitywithin 12 hours after post-infusion. Most tissues reached maximumconcentration (C_(max)) within 24 h after the dose. Recoveredradioactivity after 5 days was <5% of the dose in any single tissue. By70 days after the dose, only the carcass retained >1% (2.34%) of theadministered radioactivity. Thus, dalbavancin did not accumulate in anysingle tissue, organ, or blood cellular component. Concentrations ofradioactivity in the CNS were low but detectable in this healthy animalmodel. Dalbavancin was found to penetrate the skin with concentrationsof drug-derived radioactivity that were as high as or higher than inplasma. Blood to plasma ratio of drug-derived radioactivity remainedrelatively constant over time and was <1.

As part of the QTD studies, bile samples were collected from bile ductcannulated rats (4 animals) through 384 h (16 days) post-dose. Almost11% of the dose was recovered in the bile over 384 h after the dose.This represents the majority of the drug-derived radioactivity found infeces.

Example 9 Pharmacodynamic Activity of Dalbavancin

The goal of antimicrobial therapy is to provide active concentrations atthe site of infection for an adequate period of time to eradicateinvading pathogens. The primary method for assessing antimicrobialactivity in vitro is determination of the minimum inhibitory andbactericidal concentrations (MIC and MBC). However, these parametersonly measure net effects for a prescribed incubation period and do notcharacterize the time course of antimicrobial activity. The MBC does notdetermine if the rate and extent of bactericidal activity can beenhanced by increasing concentrations of drug. Furthermore, MICdeterminations do not measure whether there are inhibitory effects onbacteria that persist after drug removal.

An increasing number of studies suggest that the rate of bactericidalactivity, its dependence on concentration or the time of exposure, andthe presence or absence of a postantibiotic effect more clearly describethe time course of antimicrobial activity and are importantpharmacodynamic parameters for determining optimal dosing regimens. Forexample, bacterial killing by beta-lactam antibiotics shows littleconcentration dependence and the extent of killing is due primarily tothe duration of exposure. In addition, beta-lactams produce short or nopostantibiotic effects (PAE) with gram-negative bacilli. Thus, one wouldpredict that dosing regimens that maintain drug levels above the MIC fora sufficient period of time would demonstrate the best efficacy. Thishas been confirmed in several animal models. On the other hand,fluoroquinolones exhibit concentration-dependent killing and produceprolonged in-vivo postantibiotic effects. One would predict that theamount of drug rather than the frequency of dosing would be theimportant determinant of efficacy for these drugs. This has also beenconfirmed in several animal models. More importantly, the magnitude ofthe pharmacokinetic/pharmacodynamic parameter required for efficacy forboth beta-lactams and fluoroquinolones has been similar in animalinfection models and human infections.

The following studies were designed to characterize the in-vivopharmacodynamic characteristics of dalbavancin. The impact of the dosingregimen on the in vivo efficacy of dalbavancin in the experimental thighinfection model in neutropenic mice was determined. Studies were alsoperformed to investigate [1] which pharmacokinetic parameter (peak serumlevel, area under the concentration-versus-time curve (AUC), theduration of time serum levels exceed the MIC) best predicts efficacy ofdalbavancin and [2] whether the magnitude of the PK/PD parameterrequired for efficacy is similar among common gram-positive bacteriaincluding penicillin-resistant pneumococci and methicillin-resistant S.aureus. Lastly, the effect of infection site on the activity ofdalbavancin against both S. pneumoniae and S. aureus in both the thighand pneumonia infection models was determined.

Study Organisms and MICs to Dalbavancin

The study organisms and their MICs to dalbavancin are listed in Table29. MICs were determined in MHB by standard NCCLS microdilutiontechniques. MHB was supplemented with 3% lysed horse blood for MICdeterminations with S. pneumoniae. All MICs were performed at least induplicate. TABLE 29 In Vitro Activity of Dalbavancin Against S.pneumoniae and S. Aureus Strains. Dalbavancin Penicillin MethicillinOrganism MIC/MBC (mg/L) MIC (mg/L) MIC (mg/L) S. pneumoniae strain  11990.004 1.0 —  1293 0.004 2.0 —  1325 0.008 2.0 —  1329 0.008 2.0 — 108130.03  0.008 — S. aureus strain 25923 0.12/0.50 — 0.12 33591 0.12/0.25— >8.0 31005 0.06/0.25 — 0.12 MRSA 0.12/0.25 — >8.0 Smith 0.06/0.50 —0.12 307109  0.06/0.12 — >8.0

Five pneumococcal and six staphylococcal organisms were utilized. Thedalbavancin MICs for the pneumococci ranged from 0.004 to 0.03 mg/L. Therange of MICs for the S. aureus isolates were more narrow and higherthan those against pneumococci, ranging from 0.06-0.12 mg/L.

Pharmacokinetics

The plasma pharmacokinetics of dalbavancin in thigh-infected neutropenicSwiss ICR mice are shown in FIG. 44. Neutropenia in this and all otherstudies was produced by injections of cyclophosphamide, 150 mg/kg 4 daysprior to study, 100 mg/kg 1 day prior to study and 100 mg/kg every 48 hafter start of infection until end of study. Neutrophil counts remainedless than 100/mm³ for the duration of study. Doses of 2.5, 5, 10, 20;40, and 80 mg/kg were studied. Drug was administered by intraperitonealinjection of 0.2 ml volume. Blood was removed from groups of three miceby retroorbital aspiration into heparinized capillary tubes at 0.5, 1,2, 4, 6, 24, 48, 72, and 96 hours after dosing. Plasma was separated,and dalbavancin plasma concentrations were measured using amicrobiologic assay with Bacilus subtilus as the test organism. Peaklevels were observed by 4-6 hr. The half-lives of dalbavancin weredetermined by linear least-squares regression. AUC was calculated by thetrapezoidal rule from mean concentrations. The AUC was estimated at 24,36, 48, 72, 96 hr and extrapolated to infinity. The 24 h AUC wascalculated as the 6-day AUC divided by 6. Dalbavancin exhibited linearpharmacokinetics. The half-life was prolonged and varied from 7.6 to13.1 hours.

Protein Binding

The impact of drug binding to serum and serum proteins was investigatedby comparing the MIC of dalbavancin for two strains of S. aureus inbroth, infected mouse serum human serum, and albumin (Table 30). MICsfor both strains in broth was 0.12 mg/L. The MICs (arithmetic) in 95%mouse serum increased to 32 mg/L. MICs in mouse serum ultrafiltrate roseto only 0.5 mg/L suggesting that the great majority of the difference inthe MICs was do to protein binding. Similar study with human serum andalbumin resulted in an increase to only 8 mg/L. Based upon the MICdifference between broth and mouse serum, the degree of protein bindingwas estimated to be 99.6%. This degree of binding was considered insubsequent pharmacodynamic analyses. The degree of binding in humanserum would be estimated to be 96%. TABLE 30 Impact of Serum, SerumUltrafiltrate, and Human albumin on the In Vitro Activity of DalbavancinAgainst Selected S. aureus Strains. MIC 95% MIC 95% MIC 95% MIC 95%Mouse MIC Mouse Human Human Serum Organism Broth Serum Serum AlbuminUltrafiltrate S. aureus strain 25923 0.12 32 8.0 8.0 0.5 MRSA 0.12 328.0 8.0 0.5Infection Models

The murine thigh-infection model was used for all organisms throughoutthe various studies. In this well-established model, approximately 10⁶cfu of the study organisms are injected into one or both thighs (in 0.1ml) two hours before starting therapy. The number of organisms in thethigh at the start of therapy in the following studies varied from10^(7.15-7.59) cfu/thigh. The murine lung-infection model was used onlywith a single isolate of either S. pneumoniae or S. aureus. In thismodel, mice are infected by a intranasal inoculation of (approximately10^(8.5) cfu/ml) of 50 ul via the nares of anesthetized mice. Treatmentbegins 2-hrs after inoculation at which time the mice had 10^(7.4-7-6)cfu/lung.

In Vivo Time Kill

The effect of a single doses of dalbavancin on the in-vivo killing of astrain of S. pneumoniae and S. aureus over time are shown in FIGS. 45and 46. Each point represents the mean of 4 thighs. Five dose levels,ranging 16-fold were used. The dose levels used in the study against &aureus ranged from 5 to 80 mg/kg. The dose levels used in study againstS. pneumoniae ranged from 0.625 to 10 mg/kg. The extent of killing bothspecies was extensive (>2 log with the highest doses studied). However,the extent and rate of killing of the pneumococcal isolate was greaterthan the staphylococcal strain. Study with the two highest doses ofdalbavancin resulted in killing of S. aureus. Three of the five doselevels used in study against S. pneumoniae reduced the burden oforganisms nearly four log in the thighs of infected mice

Dosing Regimen Studies

In these studies, multiple dosing regimens varying the dose and dosageinterval were administered intraperitoneally in 0.2 ml volumes to groupsof mice for 144 h (6 days). The dosing intervals chosen were 12, 24, 36,and 72 hours. Five different doses (two-fold increases) were used. Studyagainst S. aureus utilized total dose (mg/kg/6 d) levels ranging from 30to 480 mg/kg/6 d. Study against S. pneumoniae utilized total dose levels(mg/kg/6 d) ranging from 0.6125 to 10 mg/kg/6 d. FIGS. 47 and 48illustrate the dose-response curves for dalbavancin at the differentdosing intervals for the strain of S. pneumoniae and S. aureus in thethighs of neutropenic mice. Each point represents the mean of 4 thighs.In general, increasing the dosing interval resulted in a slight shift ofthe dose response curves to the left indicating more efficacy with theregimens for which large doses were administered infrequently.

Each of the dose-response curves was also mathematically characterizedusing a maximum effect model. This methodology uses the Hill equation toestimate by non-linear regression the maximum effect (Emax), the dose(P50) required to obtain 50% of the Emax, and the slope of thedose-response relationship. From these parameters we could thencalculate the dose required to produce a net bacteriostatic effect overthe 144 hour treatment period as well as the doses necessary to producea 1 and 2 log reduction in organism burden. The static dose and the doseassociated with a 1 and 2 log kill for each of the drug organismcombinations and the various dosing regimens are shown in Table 31.TABLE 31 Dalbavancin Dosages Required to Achieve a Net Static Effect, 1and 2 Log Kill for Four Different Dosing Intervals in a S. pneumoniaeand S. aureus Infection Model. q 24 h q 12 h 2 log SD 1 log kill 2 logkill SD 1 log kill kill mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Organism(95% CI) 95% CI) (95% CI) (95% CI) (95% CI) (95% CI) SP10813 2.3 2.8 3.51.34 1.54 1.75 (1.9-2.7) (2.3-3.3) (2.8-4.2) (1.14-1.57) (1.34-1.74)(1.47-2.03) SA29213 80 — — 26 267 —  (8-152) (−39-91)   (−400-934)   q36 h q 72 h SD 1 log kill 2 log kill SD 1 log kill 2 log kill mg/kgmg/kg mg/kg mg/kg mg/kg mg/kg Organism (95% CI) (95% CI) (95% CI) (95%CI) (95% CI) (95% CI) SP10813 1.26 1.53 1.81 0.71 0.77 0.82 (1.16-1.36)(1.41-1.65) (1.67-1.95) (.64-.88) (.59-.95)  (.62-1.02) SA29213 15 36122 10 31 128 (−14-44)   (−34-106) (−117-361)   (−18-38)   (−55-117)(−230-486)  dose presented as total dose over 6 days (mg/kg).

Increasing the dosage regimen from 12 to 36 hours in study against S.pneumoniae did not result in an appreciable change in the dosesassociated with the three microbiologic endpoints. However, efficacywith the 72 h dosing interval required less drug. A similar relationshipwas observed in study against S. aureus. The only dosing regimens thatproduced a 1 and 2 log kill against S. aureus were the 36 and 72 hintervals.

Pharmacodynamic Parameters Correlating with Efficacy

We determined which PK/PD parameter correlated best with efficacy byrelating the number of bacteria in thigh at the end of 144 hours oftherapy with [1] the peak/MIC ratio, [2] the AUC/MIC ratio, and [3] thepercentage of the dosing interval that serum levels exceed the MIC foreach of the dosage regimens studied. The PK/PD parameter values forthose doses not specifically studied were extrapolated from the valuesof the nearest studied doses. The relationship between log cfu per thighand the peak/MIC ratio, the 24-hour AUC/MIC ratio and the percentage oftime serum levels exceeded the MIC are illustrated in FIG. 49 for S.pneumoniae and FIG. 50 for S. aureus. Each point represents the mean offour thighs. For both organisms a strong correlation was observed withthe 24-hr AUC/MIC and peak/MIC ratios. However, regression of the datawith the 24 h AUC/MIC ratio resulted in the strongest correlation. Thedata illustrated in these figures were also analyzed by the same maximumdose-response model mentioned above, except that the different PK/PDparameters were used in place of the dose. The R² represents thecoefficient of determination observed for the relationship betweenefficacy and each PK/PD parameter. The coefficient of determination (orR²) represents the percentage of the variance in bacterial numbers thatcan be attributed to each PK/PD parameter and was high for both AUC/MICand Cmax/MIC.

PK/PD Parameter Magnitude or Target

To determine if the AUC/MIC required for a static effect was similar formultiple pathogens, we studied the in vivo activity of 24- and 72-hourlydosing regimens of dalbavancin against 5 strains of S. pneumoniae and 6strains of S. aureus after 6 days of therapy. The dose-response curvesfor dalbavancin against these various strains are shown in FIGS. 51 and52. In FIGS. 51 and 52 the dose is represented by the free drug mean 24h AUC/MIC ratio over the 6 day period of study. In general, the shape ofthe dose-response curves was similar for all strains. The location ofthe dose-response curve was related to the MIC of the organism. However,the dose response curves for the pneumococcal organisms are shiftedslightly to the left. This curve shift suggests less drug was necessaryfor effect against pneumococci than staphylococci. The static dose, 1log and 2 log kill and the associated free drug 24-hr AUC/MIC andCmax/MIC are shown in Table 32. The extent of bacterial killing wasrelatively similar for most strains. All strains exhibited more than a 4log₁₀ drop in cfu over the 6 day study. TABLE 32 Dalbavancin EfficacyAgainst S. pneumoniae and S. aureus. Static Effect 1 Log Kill 2 Log KillMIC/MBC f24AUC/ f24AUC/ mg/kg/ f24AUC/ f24AUC/ mg/kg/ f24AUC/ f24AUC/Organism (mg/L) MIC MBC 24 h MIC MBC 24 h MIC MBC q 24 h mg/kg/ regimen24 h SP1199 0.004 0.40 16.0 0.49 19.4 0.57 22.7 SP1293 0.004 0.44 17.40.55 21.7 0.65 25.9 SP1325 0.008 1.46 29.0 1.6 31.8 1.73 34.5 SP13290.008 0.90 18.0 1.18 23.5 1.45 26.7 SP10813 0.03  1.34 7.5 1.54 8.7 1.759.9 Mean ± SD 0.91 ± 0.44 17.6 ± 6.9   1.1 ± 0.47  21 ± 7.4 1.23 ± 0.5224.3 ± 8.2  SA25923 0.12/0.25 42.7 216 52 51.2 250 60 60 289 69.3SA33591 0.12/0.25 22.3 96.2 46 27.7 121 58.3 33.5 157 74.3 SA310050.06/0.12 49.3 483 242 — — — — — — MRSA 0.06/0.25 37.7 374 90 45.3 452109 53.5 519 125 SA 0.12/0.25 33.5 156 75 50.7 248 119 73.5 361 173Smith Mean ± SD 37.1 ± 9.1  265 ± 143 101 ± 72  43.7 ± 9.5  268 ± 11986.6 ± 27.6 55 ± 14 332 ± 131 110 ± 42  q 72 h mg/kg/ regimen 72 hSP1325 0.008 0.83 6.0 0.99 8.8 1.16 17.6 SP1293 0.004 1.01 14.1 1.28 181.68 23.8 SP1199 0.004 0.72 10.3 0.85 12.1 0.99 35.3 SP1396 0.008 0.524.0 0.61 4.3 0.69 4.8 SP10813 0.03  0.71 1.4 0.77 1.6 0.82 1.6 Mean ± SD0.77 ± 0.18 7.2 ± 4.5 0.93 ± 0.24 9.0 ± 5.8 1.13 ± 0.36 16.6 ± 12.3SA25923 0.12/0.50 160 274 66 185 317 76.1 214 367 88.1 SA33591 0.12/0.2574 123 59 93 160 76.6 114 195 94 MRSA 0.06/0.25 43 147 35.3 62 202 48.885 292 70 SA 0.12/0.25 59 96 46.1 95 163 78.0 148 254 22 Smith SA3071090.06/0.50 31 94 11.3 34 108 12.9 37 121 14.6 SA31005 0.06/0.12 67.7 223112 94.6 325 163 127 364 217 Mean ± SD 72 ± 41 160 ± 67  55 ± 31 94 ± 46 213 ± 8.14 75.9 ± 45.2 120 ± 54  266 ± 88  101 ± 61 

For the dalbavancin regimens utilizing every 24 h dosing, the free drug24 h AUC/MIC value associated with a static effect against S. pneumoniaeand S. aureus were 17.6±6.9 and 265±143, respectively. The dose responsecurves were steep and the 24 h AUC/MIC values associated with a 1 and 2log kill were not appreciably higher. Therapy against pneumococci, basedupon the 24 h AUC/MIC, required 12 to 23 fold less drug, as suggested bythe dose response curves. For S. aureus the MBC was 2 to 4-fold higherthan the MIC. If one considers the AUC/MBC for S. aureus, the valueswould be only 4 to 8-fold higher than for S. pneumoniae.

For the regimens that utilized less frequent dosing (every 72 h), thefree drug 24 h AUC/MIC values associated with a static effect against S.pneumoniae and S. aureus were 7.2±4.5 and 160±67, respectively. ThePK/PD magnitudes necessary to achieve the three in vivo microbiologicendpoints (static dose, 1 and 2 log kill) were lower for the more widelyspaced dosing regimens. When dalbavancin was dosed every 72 h, the 24 hAUC/MIC values associated with the various endpoints were 1.3 to 2.4fold lower than when drug was dosed every 24 h.

Penicillin-resistance in S. pneumoniae and methicillin-resistance in S.aureus did not impact the 24 h AUC/MIC required for dalbavancinefficacy.

Impact of Neutrophils on Activity of Dalbavancin

To determine the effect of neutrophils on the activity of dalbavancin,we compared the dose-response curves with 24-hour dosing of the drug inboth normal and neutropenic mice infected with S. pneumoniae. Thesubsequent dose response curves are shown in FIG. 53. Each pointrepresents the mean value from 4 thighs. The static dose, and the dosesassociated with a 1 and 2 log kill were calculated from the parametersestimated by non-linear regression using the Hill equation. The doses(mg/kg/6 d) required to achieve these endpoints in both normal andneutropenic mice are shown in Table 33. The presence of neutrophilsresulted in a 1.7- to 2.1-fold reduction in the doses necessary forefficacy. However, these differences were not statistically significant.TABLE 33 Impact of Neutrophils on the In Vivo Efficacy of DalbavancinAgainst S. pneumoniae. 1 log kill mg/kg 2 log kill mg/kg SD mg/kg (95%Cl) (95% Cl) (95% Cl) Normal 2.03 (1.99-2.07) 2.35 (2.26-2.44) 2.66(2.56-2.76) Neutropenic 4.13 (0.23-8.0)  4.38 (0.28-8.50) 4.62(0.32-8.90)Impact of Infection Site on Activity of Dalbavancin

To determine the impact of infection site on the activity ofdalbavancin, we compared the dose-response curves with the 24-hourdosing of the drug in both the thigh and lung infection models (FIG.54). Both S. pneumoniae and S. aureus were utilized in these models. Thedose-response curves in the two models utilizing S. pneumoniae werenearly identical. In similar study against S. aureus the dose responsecurve in the lung model is shifted to the left suggesting less drug wasnecessary for efficacy at this site of infection. However, theconfidence intervals in the staphylococcal study were large and thesedifferences were not significant.

Conclusions

The above studies have characterized the in-vivo pharmacodynamicactivity of dalbavancin against a variety of pathogens. These can besummarized as follows.

-   -   Dalbavancin produced in vivo bactericidal activity against        both S. pneumoniae and S. aureus in both the thigh and lung        infection modes.    -   The efficacy of dalbavancin was dose-dependent.    -   The dose-dependent PK/PD parameters, 24 h AUC/MIC and Cmax/MIC,        were strongly correlated with the in vivo activity of        dalbavancin. Regression of the dose-response data with the 24 h        AUC/MIC parameter resulted in the highest coefficients of        determination. Therapy with the most widely spaced dosing        intervals was more effective in these models.    -   The 24 h AUC/MIC values associated with microbiologic effect        were similar among the strains within the species studied.        Beta-lactam resistance in S. pneumoniae and S. aureus did not        impact dalbavancin in vivo activity.    -   Efficacy against S. pneumoniae required lower 24 h AUC/MIC        values than against S. aureus. This may in part be explained by        the species differences in MIC and MBC.    -   Efficacy against S. pneumoniae, whether based upon a net static        effect, 1 or 2 log kill required a free drug AUC/MIC over the 6        days of therapy near 100 in these infection models. Efficacy        against S. aureus, similarly based, required a free drug AUC/MIC        over the 6 days of near 1000.    -   Neutropenia had minimal impact on in vivo dalbavancin activity.

Example 10 Quantitative Determination of Dalbavancin in Plasma byHPLC-MS/MS

A HPLC-MS/MS method was developed for quantitative measurement ofdalbavancin in plasma, as described below.

Preparation of Dalbavancin Calibration and Quality Control Standards

Stock solutions of dalbavancin were prepared by dissolving dalbavancinin deionized water to prepare a 1000 μg/ml solution, followed by serialdilutions in deionized water to prepare 500, 50 and 10 μg/ml solutions.

Calibration standards of 100, 60, and 40 μg/ml dalbavancin concentrationwere prepared by spiking human plasma with appropriate volumes of a 1000μg/ml dalbavancin stock solution prepared as described above.Calibration standards of 20 and 10 μg/ml concentration were prepared byspiking human plasma with appropriate volumes of a 500 μg/ml dalbavancinstock solution, and a calibration standard of 0.5 μg/ml was prepared byspiking human plasma with an appropriate volume of a 10 μg/ml stocksolution.

Quality control standards of 90 and 30 μg/ml dalbavancin were preparedby spiking human plasma with an appropriate volume of a 1000 μg/mldalbavancin stock solution prepared as described above. A qualitycontrol standard of 1.5 μg/ml was prepared by spiking human plasma withan appropriate volume of a 50 μg/ml solution.

Preparation of Internal Standard Working Solution

A 30 μg/ml working solution of internal standard BI-K0098, which is thediethyl-amino-propyl-amino derivative of A-40926, was prepared asfollows. Approximately 10 mg of BI-K0098 was dissolved in approximately10 ml of mobile phase A (80% of 10 mM Ammonium Formiate/Formic Acid, pH3 (v/v), 10% of Acetonitrile (v/v), and 10% 2-Propanol (v/v)) to make a1000 μg/ml internal standard stock solution. The stock solution (300 μl)was then diluted to a volume of 10 ml with mobile phase A to make a 30μg/ml internal standard solution.

Preparation of Samples for Analysis

Samples were prepared as follows for quantitative determination ofdalbavancin concentration in plasma. To 50 μl of calibration or qualitycontrol standards prepared as described above, 100 μl of internalstandard working standard solution was added and mixed. The mixture waspermitted to equilibrate for five minutes at room temperature, followedby addition of 250 μl of acetonitrile. The mixture was then vortexed for10 seconds, followed by centrifugation for 1 minute at about 10,000 rpmon an ALC micro-centrifugette 4214. Supernatants were transferred toclean tubes and evaporated to dryness in a Savant Speed-Vac System atabout 40° C. Samples were then resuspended in 150 μl of mobile phase A.

Analytical Method

50 μl samples prepared for analysis as described above were injectedinto a Phenomenex Jupiter C₁₈ column (50×2 mm, C₁₈ 5 μm 300 A), andanalyzed under gradient HPLC conditions at a flow rate of 0.3 ml/min.The gradient conditions were: initial, 80% mobile phase A/20% mobilephase B (20% 10 mM Ammonium Formiate/Formic Acid, pH 3 (v/v), 40%Acetonitrile (v/v), 40% 2-propanol (v/v)); 1 minute, 20% mobile phaseA/80% mobile phase B; 2 minutes, 20% mobile phase A/80% mobile phase B;2.5 minutes, back to initial conditions.

The HPLC system was coupled to a PE SCIEX API-2000 triple quadrupolemass spectrometer, with turbo ion spray operating in a positiveionization mode. Air was used to generate a spray in the ion source.Probe temperature was set at 500° C. with nitrogen as curtain gas.Multiple reactions monitoring (MRM) was employed using nitrogen ascollision gas. The analytes were detected by monitoring the followingion transitions: 909.3 Da→1429.3 Da for dalbavancin, and 923.3 Da 1457.3Da for the internal standard (BI-K0098). To avoid mass spectrometercontamination, a post-column flow diversion in the first minute and 2.5minutes after the beginning of the chromatographic run was performed.

Software Sample Control 1.4 was used for the acquisition of dataanalysis and software MacQuan 1.6 was used for the integration ofchromatographic peaks and statistical data evaluation.

Calibration Curves

Linearity of the assay method was assessed by assaying calibrationstandards to generate a calibration curve. The concentration ofdalbavancin in a plasma sample was determined by calculating the peakarea ratio between dalbavancin and the internal standard.

Calibration curves for dalbavancin concentrations over an analyticalrange of 0.5-100 μg dalbavancin/ml of human plasma were constructedusing the equation y=A+Bx (weighted 1/x), where A represents interceptof the curve, B represents the slope of the curve, x represents thedalbavancin concentration of calibration standard (μg/ml), and yrepresents the peak area ratio of dalbavancin to internal standard.Three separate calibration curves were constructed. The results showedthat dalbavancin/internal standard area ratio and dalbavancinconcentrations varied linearly over the analytical range. The lowerlimit of quantitation (LLOQ) was 0.5 μg dalbavancin per ml of humanplasma. The slopes for the calibration curves were reproducible andtheir correlation coefficients were greater than 0.9995.

Stability of Dalbavancin in Plasma

The stability of dalbavancin in plasma samples was tested by analyzingthree replicates quality control standards of human plasma samples,prepared as described above, at two different concentrations, 1.5 and 90μg/ml. Detectable dalbavancin concentration was stable after threecycles of freeze-thaw treatment. Dalbavancin concentration in processedsamples was stable after 24 hours at room temperature. No reduction indalbavancin concentration with respect to time zero samples wasobserved.

Example 11 Dalbavancin Mass Spectroscopy Analysis

The nature of dalbavancin multimers in solution was investigated and theconditions influencing the population ratio of dalbavancin multimer todalbavancin monomer were determined by electrospray ion massspectroscopy (ESI-MS).

Experiments were performed using an Applied Biosystem API III+ massspectrometer equipped with a TurbolonSpray source, a Triple Quadrupoleanalyzer, operating in positive ion mode. The optimized conditions arereported in Table 34 below. TABLE 34 Instrumental Conditions forDalbavancin Analysis on Applied Biosystem API III+ Mass Spectrometer.IonSpray source: IonSpray voltage 5000 V Orifice plate voltage 80 VCurtain gas flow 0.6 L/min Nebulizer gas flow 1.2 L/min Liquid flow 5μL/min Interface heater 60° C. Scan conditions (Q1 scan): Step 0.1 amuDwell time 1 msec MS analyzer: Interface plate voltage 650 V Q0 roadoffset voltage 40 V Q1 park mass 1000 Q1 resolution 120.8 Q1 delta mass0.2 Q1 road offset voltage 27 V Lens 7 voltage −50 V Q2 road offsetvoltage −50 V Q3 park mass 1000 Q3 resolution 110 Q3 delta mass 0 Q3road offset voltage −70 V Lens 9 voltage −250 V Faraday plate voltage−250 V Channel electron multiplier −3800 V voltageDalbavancin in Solution

Instrumental parameters were tuned on a dalbavancin solution containing0.1 mg/ml of dalbavancin dissolved in a 8:2 water:isopropanol solution.A spectrum of dalbavancin in solution was acquired in the range of500-2000 amu following direct injection of the solution. The resultingspectrum, as seen in FIG. 7, indicates the presence of dalbavancinmultimers. As a non-limiting example, one trace of the spectrum isattributable to a homomultimer of B₀, which is present as a (2nM+y(⁺3))ion species, where n is a positive integer indicating the multiplicityof the homomultimer, e.g., n=1 when the multimer is a homodimer and n=2when the multimer is a homotetramer, M indicates the mass of themonomer, y=n and ⁺3 indicates a plus three ion charge. For example, thehomodimer of B₀ is provided when n=1, y=1, and M=mass of B₀. Thishomodimer species is assigned to a (2M ⁺3) ion trace in the massspectrum.

Influence of Dalbavancin Concentration on the Population Ratio ofDalbavancin Multimer to Monomer

The influence of dalbavancin concentration on the population ratio ofmultimer to monomer was evaluated by mass spectroscopy using theconditions described above. The spectra were acquired by direct infusionof dalbavancin solutions at concentrations of 20, 40, 60, and 80 μg/mL.The intensities of the main peaks were reported as a function ofdalbavancin concentration and the population ratios of dalbavancinmultimer to monomer were determined, as shown in FIG. 8.

The data indicate that the population ratio of dalbavancin multimer todalbavancin monomer increases with increasing concentration. This mayhelp to explain the high drug loading capacities that may beadministered to an individual. The role of multimer as a depot ofmonomer may decrease the tendency of higher concentration samples toform precipitates and enhance the concentrations which may beadministered to an individual. The presence of multimers may also allowrapid administration of a dose of dalbavancin to an individual.

A non-limiting example of a method of determining the population ratioof dalbavancin multimer to monomer is provided, for example, bydetermining the ratio between peak intensities of ions A and B as shownin FIG. 7. Dividing the intensity of peak A by the intensity of peak Bprovides one measure of the population ratio of dalbavancin multimer tomonomer.

Influence of pH on the Population Ratio of Dalbavancin Multimer toMonomer

The influence of solution pH on the population ratio of dalbavancinmultimer to monomer was evaluated at the instrumental conditionsdescribed above and at the following solution pH values: 2.5, 3.0, 3.5,4.0, 4.5, 5.0, and 5.5. The population ratio of dalbavancin multimer tomonomer was determined at each of the pH values and plotted against pH,as seen in FIG. 9. It was determined that the population ratio ofdalbavancin multimer to monomer increases with increasing pH.

While not to be limited to theory, it is believed that ionic groups,such as a carboxylate group on a first dalbavancin monomer, aid in thestabilization of dalbavancin multimers by forming ionic interactionswith oppositely charged ions, such as tertiary nitrogen groups, on asecond dalbavancin monomer. Such ionic interactions can be influenced bypH. It is believed that the increasing tendency of dalbavancin to bepresent as a multimer at higher pH is indication that ionic interactionsare important in multimer stabilization. In particular, it is believedthat dalbavancin multimers are destabilized at lower pH, presumably dueto interruption of the ionic interactions contributing to multimerstability as certain functional groups, such as carboxylate groups, maybe protonated at lower pH values.

Influence of Solution Ionic Strength on the Population Ratio ofDalbavancin Multimer to Monomer

The influence of solution ionic strength on the population ratio ofdalbavancin multimer to monomer was determined by mass spectrometry. Themass spectra were obtained in electrospray positive mode on a FinniganLCQ^(Deca) ion trap instrument previously tuned and calibrated inelectrospray mode using Ultramark 1621, caffeine and MRFA(L-metionyl-arginyl-phenilalanyl-arginine). All the mass spectra wererecorded using the conditions listed in Table 35. The sample parametersthat were investigated are listed in Table 36. TABLE 35 MS ConditionsSample Inlet Conditions: Capillary Temperature (° C.): 200 Sheat Gas(N_(2,) arbitrary units): 40 Sample Inlet Voltage Settings: Polarity:positive Source Voltage (kV): 4.70 Capillary Voltage (V): 34 Tube LensOffset (V): −60 Full Scan conditions: Scan range (amu):  500-2000 Numberof microscans: 3 Maximum ion time (ms): 200 Zoom Scan conditions: Scanrange (amu): 1218-1228 Number of microscans: 5 Maximum ion time (ms): 50

TABLE 36 Sample Parameters Sample μg/mL Solvent pH Dalbavancin 100COO⁻NH₄ ⁺ 5 mM 5 100 COO⁻NH₄ ⁺ 50 mM 5 100 COO⁻NH₄ ⁺ 100 mM 5

Sample water solutions were infused at 10 μL/min via a Harward syringepump and the mass spectra were obtained as seen in FIGS. 10-12.

The obtained spectra indicate that the population ratio of dalbavancinmultimer to monomer is influenced by ionic strength. An increase inbuffer concentration was found to correspond to a decrease in multimermass traces and hence a decrease in dalbavancin multimer to monomerpopulation ratio.

As mentioned, it is believed that ionic interactions are important indalbavancin multimer stability. The fact that increasing ionic strengthwas correlated with decreasing intensity of multimer mass tracessubstantiates the role of ionic interactions in multimer stability.However, as multimer mass traces were present even at higher ionicstrengths, another, second, interaction may be involved in multimerstabilization.

While not bound to any theory, it is believed that hydrophobicinteractions are important in stabilizing the multimer species ofdalbavancin. If the stabilization of these non-covalent dalbavancinmultimers was solely due to ionic interactions, it would be expectedthat an increase in ionic strength would result in total loss ofmultimer mass species. That is, it would be expected that as the ionicstrength of the solution increases, the ionic interactions stabilizingthe multimer would be disrupted by the increased population of ions insolution, with which the monomers would more readily associate.Consequently, the solution ionic strength would drive the multimers todisassociate into monomer components and the resulting mass spectrawould be free of any multimer mass traces. However, even at highsolution ionic strength (such as 100 mM ammonium formate), the presenceof dalbavancin multimers is detected in the mass spectrum. Accordingly,the multimers of dalbavancin are deemed to be stabilized, at least inpart, by hydrophobic interactions.

Structurally Similar Compound

It is believed that the improved efficacy of Dalbavancin is due at leastin part to its ability to form multimers. It is thought that this uniquecharacteristic is not shared even by very structurally similarcompounds. A compound with a chemical structure similar to Dalbavancinwas investigated by mass spectroscopy analysis for its ability to formmultimers. The mass spectra were obtained in electrospray positive modeon a Finnigan LCQ^(Deca) ion trap instrument previous tuned andcalibrated in electrospray mode using Ultramark 1621, caffeine and MRFA(L-metionyl-arginyl-phenilalanyl-arginine). All the mass spectra wererecorded using the conditions listed in Table 37. The sample parametersthat were investigated are listed in Table 38. Sample water solutionswere infused at 10 μL/min via a Harward syringe pump and mass spectrawere obtained as seen in FIGS. 13 and 14. TABLE 37 Mass SpectraConditions Sample Inlet Conditions: Capillary Temperature (° C.): 200Sheat Gas (N_(2,) arbitrary units): 40 Sample Inlet Voltage Settings:Polarity: positive Source Voltage (kV): 4.70 Capillary Voltage (V): 34Tube Lens Offset (V): −60 Full Scan conditions: Scan range (amu): 500-2000 Number of microscans: 3 Maximum ion time (ms): 200 Zoom Scanconditions: Scan range (amu): 1218-1228 Number of microscans: 5 Maximumion time (ms): 50

TABLE 38 Sample Parameters Sample μg/mL Solvent pH Teicoplanin 50 H₂On.a. 100 H₂O n.a.n.a. = not adjusted

A similar glycopeptide antibiotic (teicoplanin) does not show multimericcomplexes in solution at various concentrations. This supports theindication that structurally similar compounds fail to form multimericspecies in solution, and that this phenomenon may play an important rolein the activity of the dalbavancin.

Example 12 Matrix-Assisted Laser Desorption/Ionisation Time of Flight(MALDI-TOF) Mass Spectrometry of Protein-Dalbavancin Complexes

10 μl HSA, 0.150 mM was mixed with 10 μl dalbavancin solution (from0.075 mM, 0.15 mM, 0.3 mM and 1.5 mM) and incubated for 60 min at 37° C.The samples were prepared for analysis using the dried droplettechnique. Spectra were obtained on a BRUKER FLEX III, tof massspectrometer previously tuned and calibrated using standard bovine serumalbumin, acquiring and averaging spectra generated by 200 laser shots.Matrix: 9 parts of DHB-9 (2,5-dihydroxy-benzoic acid) saturated inacetonitrile/H₂O (50:50), 1 part of sinapinic acid saturated inacetonitrile/H₂O (50:50). 0.5 ul of sample solution and 0.5 ul of matrixsolution were mixed and placed on the laser target.

Dalbavancin binds to the protein as the monomer (1HSA+1 dalbavancin). Atvery high dalbavancin to protein ratios (1:2, 1:10), the presence ofcomplexes containing 2 molecules of dalbavancin per protein molecule canbe observed.

Example 13 Isothermal Titration Calorimetry of Binding of Dalbavancin toN,N′-diacetyl-Lys-D-Ala-D-Ala in the Presence of Human Serum Albumin

The binding of dalbavancin to N,N′-diacetyl-Lys-D-Ala-D-Ala, a peptideanalog of cell-wall targets of dalbavancin, was investigated byisothermal titration calorimetry (ITC) in the presence of HSA over arange of concentrations (up to 600 μM) at 25° C., with some additionalmeasurements at 37° C. HSA increased the solubility of dalbavancin andreduced its binding affinity for the tri-peptide ligand. Results werecompared with those for vancomycin. The observed effects plateaued atrelatively low HSA concentrations, consistent with a non-competitivebinding model that allows binding of ligand to dalbavancin both free insolution and (more weakly) to the dalbavancin-HSA complex.

Preliminary experiments demonstrated that, in the absence of serumproteins, dalbavancin and vancomycin show similar binding profiles: bothgive exothermic binding to N,N′-diacetyl-Lys-D-Ala-D-Ala, but noevidence of binding to dipeptide (D-Ala-D-Ala) or toLys-D-Ala-D-Lactate. For dalbavancin/tri-peptide interaction, the datawere consistent with binding with K_(diss)=1-10 μM, depending ontemperature, similar to vancomycin under the same conditions. In thepresence of HSA, the solubility of dalbavancin is significantlyincreased and the binding affinity for tripeptide is reduced in a mannerconsistent with competitive or non-competitive binding by HSA for theantibiotic. The experiments described in this Example were designed inorder to: (a) compare dalbavancin/tri-peptide measurements at differenttemperatures (25 and 37° C.) and different HSA concentrations; (b) usethese data to construct a binding model to compare with observednumbers.

Dalbavancin was supplied by Biosearch Italia. Other reagents were fromSigma: vancomycin hydrochloride (Sigma V-2002, fw 1485.7),N,N′-diacetyl-Lys-D-Ala-D-Ala (Sigma D-9904, fw 372.4), human albumin(HSA; Sigma A-3782; mw 69,366).

Antibiotics and peptides were dissolved in aqueous buffer (20 mM Naphosphate, 150 mM NaCl, pH 7.4) containing HSA, with gentle stirringimmediately before each experiment. Peptide concentrations weredetermined by weight. Dalbavancin concentrations were determined eitherby weight or by UV absorbance using the molar extinction coefficientsε=12430 (dalbavancin, A₂₈₀ ^(1%)=68.42), ε₂₈₀=6690 (vancomycin). HSAconcentrations were determined by UV absorbance (HSA, ε₂₈₀=37,700; A₂₈₀^(1%)=5.44). Spectra were recorded at room temperature in 1 cmpathlength quartz cuvettes using Shimadzu UV-160A or UV-1601spectrophotometers, with samples diluted quantitatively with buffer ifnecessary to give absorbance in the 0.1-1 A range.

Isothermal titration calorimetry was performed using a Microcal VP-ITCinstrument at 25° C. and 37° C. using standard operating procedures.See, e.g., Wiseman et al., Anal. Biochem. (1989) 179, 131-137; Cooper,et al., Philos. Trans. R. Soc. Lond Ser. A-Math. Phys. Eng Sci. (1993)345, 23-35; Cooper, A, Isothermal Titration Microcalorimetry in C.Jones, B. Mulloy and A. H. Thomas (Eds.), Microscopy, OpticalSpectroscopy, and Macroscopic Techniques. Humana Press, Totowa, N.J.,(1994) p 137-150; Cooper, A., Microcalorimetry of Protein-proteinInteractions in J. E. Ladbury and B. Z. Chowdhry (Eds.); Biocalorimetry:The Applications of Calorimetry in the Biological Sciences. Wiley,(1998) p 103-111; and Cooper, A., Curr. Opin. Chem. Biol. (1999) 3,557-563. Samples were degassed gently prior to loading to prevent bubbleformation in the calorimeter cell. Each experiment typically comprised25×10 μl injections of peptide solution (≈1 mM) into the calorimetercell (volume≈1.4 ml) containing antibiotic solution (≈20-100 μM).Control experiments involved injection of ligand into buffer underidentical conditions to determine heats of peptide dilution, and thesevalues were used for correction of raw binding data prior to analysis.Dalbavancin/tri-peptide binding experiments were repeated several timesat each temperature. ITC binding data were analyzed using standardMicrocal ORIGIN™ software to determine the apparent number of bindingsites (N), the binding affinity (K_(ass)=1/K_(diss)) and enthalpy ofbinding (ΔH). TABLE 39 Thermodynamic data for binding of tri-peptidebinding to dalbavancin and vancomycin determined by ITC assuming asimple non-cooperative binding model: effects of temperature and HSA. T° C. N K_(ass) M⁻¹ Kdiss μM ΔH kcal/mol ΔS eu [HSA] μM Dalbavancin 100.59 6.70E+05 1.49 −10.26 −9.60 0 10 0.59 9.20E+05 1.09 −8.95 −4.30 0 100.59 6.85E+05 1.46 −10.30 −9.60 0 10 0.56 6.24E+05 1.60 −10.30 −9.90 025 0.6  3.13E+05 3.19 −10.10 −8.90 0 25 x 3.30E+05 3.03 −11.30 −12.60 025 x 3.20E+05 3.13 −11.70 −14.00 0 25 x 2.80E+05 3.57 −14.30 −23.00 0 250.57 3.03E+05 3.30 −12.60 −17.00 0 25 0.74 3.19E+05 3.13 −11.20 −12.50 025 0.54 3.93E+05 2.54 −12.90 −17.60 0 with HSA 25 0.37 1.18E+05 8.47−26.40 −65.50 13.6 with HSA 25 0.35 1.18E+05 8.47 −27.80 −70.10 13.6with HSA 25 0.68 3.50E+04 28.57 −20.00 −46.20 34.2 with HSA 25 0.832.76E+04 36.23 −16.90 −36.50 80.7 with HSA 25 1.38 3.49E+04 28.65 −18.60−41.70 104 with HSA 25 0.9  3.10E+04 32.26 −21.05 −50.00 288 with HSA 25 1.242 2.84E+04 35.21 −19.50 −44.90 430 with HSA 25 0.86 2.18E+04 45.87−15.00 −30.40 601 37 0.82 1.30E+05 7.69 −12.50 −16.80 0 37 0.6  1.10E+059.09 −17.40 −33.20 0 with HSA 37  0.179 1.25E+04 80.00 −98.60 −299.00482 with HSA 37 0.5  9568 104.52 −26.60 −67.60 516 Vancomycin 10 1.1 6.90E+05 1.45 −9.49 −6.80 0 25 0.91 3.40E+05 2.94 −10.70 −10.60 0 250.97 3.60E+05 2.78 −12.90 −17.80 0 with HSA 25 1.05 2.90E+05 3.45 −10.09−8.80 513

ITC experiments on the binding of tri-peptide to dalbavancin in theabsence of HSA give consistent data for binding affinities, with averageK_(diss) in the region of 1.4, 3.1, and 8.4 μM at 10, 25, and 37° C.respectively (Table 39). Binding is exothermic. Concentrationcalculations here indicate that N is closer to 0.5 under theseconditions. This is consistent with the binding of one tri-peptidemolecule per dalbavancin dimer under these conditions. These bindingaffinities and enthalpies of binding are comparable to those observedwith vancomycin under the same conditions (Table 39, and D. McPhail, A.Cooper, J. Chem. Soc.-Faraday Trans. (1997) 93, 2283-2289.). Note alsothat vancomycin undergoes ligand-induced dimerization at higherconcentrations.

Addition of HSA to the dalbavancin mixtures reduces the apparent bindingaffinity between tri-peptide and dalbavancin, though the binding isapparently more exothermic (Table 39). The HSA concentration dependencefor this at 25° C. is shown in FIG. 15. After an initial rise(weakening) in apparent K_(diss) up to 35 μM HSA, it remains relativelyconstant for higher HSA concentrations approaching physiological levels(600 μM). The plateau level at high concentrations of HSA (K_(diss)≈35μM) corresponds to around 10-12× weaker binding affinity than in theabsence of HSA. A similar reduction is seen at 37° C.

The HSA effect was not due to interaction with the tri-peptide. ControlITC experiments for binding of vancomycin to tri-peptide in the presenceof HSA gave results comparable to those seen in the absence of HSA (seeTable 39). This indicates that neither the peptide nor the closelyrelated antibiotic, vancomycin, interact with HSA in solution. Itfollows that any effect of HSA on dalbavancin/tri-peptide interactionmust be due to interaction between HSA and dalbavancin.

Although not wishing to be bound by theory, the above data areconsistent with a non-competitive binding model. This model assumes thatthe tri-peptide ligand (L) can bind to dalbavancin (D) both in the freestate and (possibly with different affinity) in the dalbavancin-HSAcomplex.

-   -   D+L≈DL; K_(L)=[D][L]/[DL]    -   D+HSA≈D.HSA; K_(HSA)=[D][HSA]/[D.HSA]    -   D.HSA+L≈LD.HSA; K_(LDHSA)=[D.HSA][L]/[LD.HSA]

Apparent (Observed) Ligand Binding Dissociation Constant(Non-Competitive): K_(app, L) = [total  D][L]/[total  DL  complex]$\begin{matrix}{= {{\left( {\lbrack D\rbrack + \left\lbrack {D.{HSA}} \right\rbrack} \right)\lbrack L\rbrack}/\left( {\lbrack{DL}\rbrack + \left\lbrack {{LD}.{HSA}} \right\rbrack} \right)}} \\{= {K_{L}{\left\{ {1 + {\lbrack{HSA}\rbrack/K_{HSA}}} \right\}/\left\{ {1 + {{\lbrack{HSA}\rbrack.K_{L}}/{K_{HSA}.K_{LDHSA}}}} \right\}}}} \\\quad\end{matrix}$

This shows a hyperbolic dependence of K_(app,L) on free HSAconcentration that agrees well with the observed data (FIG. 15). At high[HSA] this reaches an asymptotic (plateau) value

-   -   K_(app,L)=K_(LDHSA) (for large [HSA])

This suggests that the binding affinity for tri-peptide is around 35 μMwhen dalbavancin is bound to HSA, compared to 3 μM for free dalbavancin(25° C. figures).

Further mechanisms may be suggested in terms of whether dalbavancin isacting as a monomer or a dimer in its interactions with peptides orproteins. Direct comparison of dalbavancin with vancomycin (which showsunambiguous 1:1 binding at these low concentrations) shows that bindingis complete at lower molar ratios (lower N) for dalbavancin (FIG. 16).This is consistent with a 2:1 dalbavancin:peptide complex.

However, in the presence of HSA, the apparent N values increase (Table39), and may be more consistent with 1:1 complexation. Although notwishing to be bound by theory, the model shown in FIG. 17, showing thepossible interaction of dalbavancin monomers and dimers with tri-peptideligands and HSA, is consistent with these observations. FIG. 17A depictsdalbavancin as in monomer-dimer equilibrium in solution (predominantlyas dimer), but binding as monomer to two separate sites on HSA. This isconsistent with the N=0.5 values observed by ITC for binding of serumproteins to dalbavancin (Example 3). FIG. 17B depicts ligand binding tothe dalbavancin dimer in solution, and (more weakly) to the dalbavancinmonomers attached to HSA. This is consistent with the non-competitivebinding of dalbavancin to both tri-peptide and to HSA, with variableapparent stoichiometries.

In sum, this Example shows that HSA reduces the binding affinity ofdalbavancin for tri-peptide ligand in a manner consistent with anon-competitive mechanism and that dalbavancin bound to HSA retains itsability to bind tri-peptide ligands, albeit with reduced affinity. Theseresults are also consistent with a model in which dalbavancin is inmonomer-multimer equilibrium, predominantly multimeric in solution, withstrong affinity of multimers for peptide ligand. Dalbavancin monomers,both free and bound to serum albumin, may also bind peptides with areduced affinity.

Example 13 A-40926 and Dalbavancin Preparations

Preparation of A-40926

A-40926, the natural glycopeptide produced by fermentation, was thestarting material for producing Dalbavancin. It was produced by aNonomuria sp ATCC 39727 as a mixture of A-40926 and its acetylderivative (see U.S. Pat. No. 4,935,238 and B. Golstain et al.Antimicrobial Agent and Chemotherapy, December 1987, p. 1961-1966). Theacetyl derivative was first deacetylated to A-40926. Afterdeacetylation, A-40926 was purified by column chromatography onpolyamide as described below. The following description isrepresentative of the current production method. The amounts reportedhere are about ¼ of the amounts usually worked in an industrialpreparation.

Deacylation of A-40926

10 m³ of fermentation broth (23° C.) containing a total of about 1 g/Lof A-40926 and its acetyl derivative were adjusted, under stirring, atpH 11.4 with NaOH 30%. Stirring was continued for 6 hours, then thetemperature was reduced to 15° C. and the broth was microfiltered (KochProtosep IV Microfilter with a 0.12 m² ceramic membrane 0.1μ). Duringmicrofiltration, water was continuously added to the retentate in orderto obtain, at the end of the process, a permeate of 20-25 m³ and aretentate of 4.5-5 m³ (one half of the starting value).

The permeate, which contained A-40926, was analyzed by HPLC. When thedeacetylation was completed, the pH of the permeate solution wasadjusted at pH 7 with 30% sulfuric acid (stored at 20° C.). In thisexample, 25 m³ of filtered broth containing 6.62 Kg of A-40926 (268mg/L) was obtained. The deacetylation yield was 66.2%. If themicrofiltration process was carried out for longer time and higherextraction volume than those employed in this process, the yield can beincreased up to 90%.

Purification of A-40926 on Polyamide Column

After extraction, A-40926 contained in filtered broth was purified on apolyamide column, as described below. The amount reported in thisdescription is about 1/10 of the amount usually worked in an industrialpreparation and is representative of the current production method.

500 L of polyamide resin SC6 (from Macherey Nagel) were suspended indemineralized water and loaded into a column. The resin was thenconditioned at pH 6-6.5 by eluting the column with at least 2 BV (bedvolume) of a buffer solution prepared by dissolving 4 Kg of sodiumcarbonate in 800 L of water and adjusting the pH of the resultedsolution with acetic acid.

The A-40926 filtered broth (9000 L; assay 0.275 mg/L; A-40926 2475 g; pH6±0.2; temperature 10±3° C.) was loaded into the column at about 5 g ofactivity per liter of resin (activity/resin ratio of 5-8 g/L was usuallyused). The column was washed with the following solutions: 3 BV (1500 L)of a solution at pH 6 prepared by dissolving 7.5 Kg of sodium carbonatein 1500 L of demineralized water and adjusting the pH with acetic acid;4 BV (2000 L) of a solution at pH 8 prepared by dissolving 10 Kg ofsodium carbonate in 2000 L of demineralized water and adjusting the pHwith acetic acid; 1.5 BV (750 L) of a solution at pH 9 prepared bydissolving 4 Kg of sodium carbonate in 750 L of demineralized water andadjusting the pH with acetic acid.

A-40926 was recovered from the column by eluting with 4 BV (2000 L) of abuffer solution at pH 10 prepared by dissolving 10 Kg of sodiumcarbonate in 2000 L of demineralized water and adjusting the pH withacetic acid. The fractions containing purified A-40926 (concentration ofA-40926 greater than 0.5 g/L and HPLC area % of the main component(Bo+B₁) greater than 80%) were collected, neutralized with 1N HCl, andanalyzed by HPLC. About 2000 L of final clear solution were obtained.

The resin used for the purification was regenerated with 1.5 BV of 1:1mixture of isopropanol/5% NaOH followed by a washing with 5 BV ofdemineralized water.

A-40926 Concentration

The solution coming from the column was subject to several rounds ofdilution/concentration steps to eliminate most of the inorganic salts inthe solution. The solution was concentrated to 80 L by nanofiltrationusing a membrane with a cut-off of 250 D, diluted with 80 L ofdemineralized water, and re-concentrated at the starting volume (80 L)by nanofiltration. This operation was repeated at least 5 times. The pHof the final solution (80 L, pH 7.5) was adjusted at pH 6.3 with 23%HCl. The solution was then diluted with 80 L of acetone, and its pH wasadjusted again at pH 2.6 with 23% HCl.

Decoloring

680 g of charcoal PA 200 C (˜0.3 g/g A-40926) was added under stirringto the solution obtained in the above step (160 L). Stirring wascontinued for at least 30 minutes at room temperature, then about0.5-0.6 Kg of filter aid (DIF-BO) was added. The mixture was filteredthrough a filter cartridge. The clear solution obtained was concentratedunder vacuo (45° C.) in order to reduce the acetone below 10%. The finalvolume was about 100 L. The pH was then adjusted at 6.7 with aq. NaOH,and the concentration step was continued using the usual nanofilteruntil the A-40926 concentration was around 100 g/L. 20 L concentratedsolution was obtained (A-40926 1884 g, 94.2 g/L).

Precipitation and Drying

The previous solution was diluted under stirring with 20 L of acetone,and its pH was adjusted at 5.1 with 10% HCl. To this solution additional5 volumes of acetone (100 L) were added to complete the A-40926precipitation. If water content was not <15% at this point, additionalacetone was added. After 2 hours the suspension was centrifuged, and thesolid was washed with 3×10 L of fresh acetone. The mother liquors wereanalyzed and discharged after having ascertained the absence of product.

Solid A-40926 was dried under reduced pressure at 30-35° C. in a staticdrier until the residual acetone was below 2% and the water was lessthan 10%. The product was then sieved through a 50 mesh sieve obtaining2.08 Kg of purified A-40926 (HPLC assay 81.4%; water 6.2%; sulphatedashes 4.8%). The yield, starting from the activity loaded on the column,was 68.4%.

Synthesis of Dalbavancin

Dalbavncin (BI-397) was prepared from the natural glycopeptide A-40926through a three-step synthesis as described in Malabarba and Donadio(1999), Drugs of the Future, 24(8):839-846. Specifically, A-40926 wasfirst subject to an esterification step to make MA, which was thensubject to an amidation step to make MA-A-1. A final hydrolysis stepthen converted MA-A-1 into dalbavancin.

Esterification Step (Step 1)

The following description is representative of the current method inuse.

Preparation of H₂O₄ 96%/MeOH (Solution A)

In a 15 L round bottomed flask equipped with a mechanical stirrer and athermometer, 2.28 L of H₂SO₄ 96% (300 mL of H₂SO₄ 96% per Kg of A-40926powder) was added drop wise to 7.9 L of MeOH. An external ice bath wasused to maintain the temperature between 0 and 5° C.

Reaction Procedure

Starting material A-40926 (7.6 kg; batch 019, assay 85.09%; activity6.46 kg; 3.73 mol) was suspended in a 140 L glass-lined reactor in MeOH(46 L), and the resulting suspension was cooled at 0° C.±2° C. At thistemperature the suspension was treated with the previously preparedSolution A (H₂SO₄/MeOH). The resulting solution was stirred at 0° C. for22-26 hours while the reaction (a reaction aliquot diluted 100 timeswith 1:1 acetonitrile/water mixture) was monitored by HPLC analysisevery two hours. The esterification was considered complete when theresidual A-40926 was less than 5% and diester was not more than 10% asHPLC area %.

Ester (MA) isolation

When the reaction was complete, the mixture was cooled at −5° C. (+/−2°C.) and diluted with a same volume of cold water (54 L) maintaining thetemperature below 5° C. The product (MA) was precipitated by adjustingthe pH of the solution at 5.5 (+/−0.2) by slowly adding 10.2 L oftriethylamine (TEA). Stirring was continued for an additional hour at0-2° C., then the solid obtained was centrifuged, washed with water (10L per Kg of A-40926) and finally with MeOH (3 L of MeOH per Kg ofstarting A-40926) previously cooled at 10-15° C. Washing with water wasdone primarily to remove sulphates from MA.

Mother liquors and washings were separately analyzed and discharged ifcontained less than 1-2% of activity. The product was dried in vacuo (50mmHg) at 35-40° C. (external temperature) until the residual water wasless than 10%. 7.6 Kg of MA (5.63 kg activity, 3.23 mol) was obtained asa brownish powder.

The analysis showed the following values of HPLC area %: MA 89.8,

A-40926 3.2, Diester derivative 5.9. The HPLC assay was 74.2%, activity5.637 Kg; 3.23 mol; yield=86.5%. This material was used in the followingstep without any further purification.

Amidation Step (Step 2)

The following description is representative of the current productionmethod.

Preparation of the DMSO/HCl Mixture (Solution B)

DMSO (1.6 L) was placed into a 10 L round bottomed flask, equipped witha mechanical stirrer and a thermometer, and cooled with an ice bathbelow 10° C. HCl 37% (1 L) was then slowly added under stirringmaintaining the temperature of the mixture below 25° C.

Amidation Procedure (Production of MA-A-1)

Starting material MA 5.95 kg (assay 76.3%, KF 8.9%; 2.68 mol) was slowlydissolved under stirring in 19.2 L of 1:1 DMSO/MeOH mixture (˜1.6 L DMSOand 1.6 L MeOH per Kg of MA powder) at room temperature. After 1 hour ofstirring 709 mL of 3-(dimethylamino)-propylamine (DMEPA, MW 102.1;density=0.812 g/mL; 5.63 mol; 1.96 mols per mol of starting MA) and 325g of 1-hydroxybenzotriazole hydrate (HOBT.H₂O; MW 153.1; 2.04 mol; 0.71mol per mol of starting MA) were added to the reaction mixture. Stirringwas continued until a complete solution was obtained, then the mixturewas adjusted at pH 3-3.1 (measured after diluting an aliquot of thereaction 10 times with water) by slowly adding about 2.0 L of Solution B(DMSO/HCl).

A dicyclohexylcarbodiammide (DCC) solution, prepared by dissolving 1.03Kg of DCC (4.99 mol; MW 206.3; 1.74 mol per mol of MA) in 4.1 L of 1:1DMSO/MeOH mixture, was added to the stirred reaction mixture in 10minutes. Stirring was continued for 5 hours, then additional 51.5 g ofsolid DCC (0.25 mol) was added to the reaction mixture in order to lowerthe residual MA under 5%, maintaining the level of isoureas lower than4-5%. Isoureas are a group of by-products produced by further reactionof Dalbavancin with the excess of DCC.

Typically after 2 additional hours (7 hours total) the reaction wascompleted. At the end the mixture was diluted with water (60 L) to lowerthe DMSO concentration to 15% (v/v) and adjusted at pH 2.3 with HCl 1N(0.85 L) to destroy any residual DCC.

Hydrolysis of MA-A-1 to Dalbavancin (Step 3)

After 30 minutes the mixture was adjusted at pH 12.0-12.1 with 15% NaOH(8 L). Stirring was continued for 4 hours maintaining the mixture atthis pH with small additions of NaOH 15%. After this time the residualMA-A-1 was less than 0.2% as HPLC area %.

The mixture was then acidified at pH 3.0 with 1N HCl (19 L), and thesuspension was filtered to remove the dicyclohexylurea formed. The solidcake was washed on the filter with demineralized water (2×20 L).Washings and filtrate were gathered together, obtaining a clear solutionwhich was analyzed by HPLC. 152.8 L of solution containing 21.74 g/L ofDalbavancin (total activity 3322 g; 1.828 mol, yield=68.2%) wasobtained.

Purification of Dalbavancin

The following description is representative of the current productionmethod.

The 152.8 L of solution obtained from the hydrolysis step and containing3322 g of Dalbavancin activity was split into two parts and each one waspurified separately on the same chromatographic column containing 400 Lof polyamide. In these two purification runs the activity/resin ratiowere 4.3 and 4.0 g/L respectively.

Polyamide Column Preparation

The glass-lined column (internal diameter=40 cm, h=320 cm) containing400 L of polyamide resin was cleaned according to the resin regenerationprocedures (see below) and conditioned with 2 BV (800 L) ofdemineralized water acidified with 4 L of AcOH (pH=3.2).

Purification of the First Portion

The first portion of 76.4 L of starting solution was diluted with H₂O(56 L) in order to lower the DMSO content under 5% (v/v), and acidifiedto pH 2.78 with 1 N HCl (3.4 L). This solution was then loaded onto thecolumn at a flow rate of 150 L/h. After loading, the resin was washedwith the following solutions: 4 BV (1600 L) of H₂O acidified with AcOH(8 L), pH=3.2; 5 BV (2000 L) of AcONa 0.1 M, pH=8.2; 1 BV (400 L) of H₂Oacidified with AcOH (1 L), pH=3.2. Dalbavancin was eluted with 4 BV(2400 L) of H₂O/MeOH (8:2) acidified with AcOH (6 L), pH=3.4.

During the elution step, 22 fractions of 50-60 L each were collected andanalyzed by HPLC. Fractions from 9 to 25 (concentration of Dalbavancinhigher than 0.5 g/L and HPLC area % of (B₀+B₁)≧80%) were pooledtogether, obtaining 969 L of solution with 1.56 Kg of Dalbavancin(yield=93.9%). This solution was then concentrated by nanofiltration,obtaining 125.7 L of solution with 1.38 Kg of Dalbavancin. 850 L ofpermeate, containing 145 g of impure Dalbavancin (8.7%), wereneutralized and discharged.

Resin Regeneration

Before re-using the resin was washed with the following solutions: 2.5BV (1000 L) of 1:1 MeOH/water acidified with acetic acid (2.5 mL/L); 2.5BV (1000 L) of 1:1 0.5% NaOH/isopropanol; 10 BV (4000 L) ofdemineralized water. The resin was then re-equilibrated with BV (800 L)of water acidified with acetic acid (2.5 mL/L).

Purification of the Second Portion

The second portion of the starting solution coming from the hydrolysisstep (76.5 L) was diluted with H₂O (56 L) to lower the DMSO contentunder 5% (v/v) and acidified to pH 2.87 with 3.0 L of 1 N HCl. Theportion was then purified as previously described in the purification ofthe first portion. The pooled fractions (vol.=972 L, Dalbavancin 1.54Kg, yield=92.7%) were concentrated by nanofiltration, obtaining 133 L ofsolution with 1.46 Kg of activity. 850 L of permeate, containing 73 g ofDalbavancin (4.3%), was discharged.

The concentrated solutions coming from the two purification steps werere-analyzed and pooled together giving 258 L of solution containing 2840g of purified Dalbavancin. The purification yield was 86%. The totalyield, starting from MA, was 58.3%.

Final Polyamide Regeneration

After the second purification run polyamide was regenerated with: 2.5 BVof 1:1 MeOH-water, acidified with AcOH (2.5 L) at pH=3.4; 2.5 BV of 1:10.5% NaOH-isopropanol; 10 BV of demineralized water.

Decoloration and Precipitation of Dalbavancin

1.5 mol of 1N HCl per mol of Dalbavancin and 0.3 g of charcoal CG1 (0.85Kg, from NORIT) per gram of Dalbavancin were added to the 258 L solutionobtained above. The mixture was stirred at room temperature for at least45 min. The pH was 3.1. The suspension was then filtered on a SUPRA DISCcartridge mod. SDP-EK1 from SEITZ-SCHENK, and the cake was washed with50 L of H₂O/MeOH 8:2. The filtrate was analyzed and concentrated againby nanofiltration, using a MPS 44 membrane with a cut off of 250 D. 21.3L of concentrated solution containing 119 g/L of Dalbavancin (pH 4.1;MeOH 1.9%, GC) was obtained. 909 mL of 1 N HCl was finally added toadjust the pH at 2.63, which corresponds to the salification ratio of1.65 mol_(HCl)/mol_(Dalbavancin).

The solution (22.2 L) was poured out, under stirring, in 200 L ofacetone. The solid obtained after decanting was centrifuged and washedwith 14 L of fresh acetone. The product was then dried under reducedpressure (50 mmHg) at 35° C., maintaining the internal temperature under30° C. for 17 hours. During the drying process, 1 L of low endotoxinwater (<250 EU/mL), divided in two portions of 0.5 L each, was sprayedon the solid after three and five hours in order to remove the residualacetone that otherwise is difficult to eliminate. The product was thensieved (50 mesh), obtaining 2592 g of Dalbavancin (HPLC Assay 82.4%;water (KF) 14%; Cl⁻ 3.0%).

Example 14 Alternative Methods for A-40926 and Dalbavancin Preparation

The methods described below are alternative methods that can used in theA-40926 and Dalbavancin preparation process.

A-40926 Preparation on XAD-7 HP

Deacetylation and Mycelium Microfiltration

150 L of fermentation broth containing A-40926 (pH 7) were stirred in asuitable reactor at room temperature (24° C.), and adjusted at pH 11.5with a 2.5 N NaOH solution (2.5 L). After 4 hours of stirring, the brothwas adjusted at pH 10.6 with 15% HCl, and microfiltered through a 0.2micron membrane. 439 L of clear permeate was collected and thenconcentrated by nanofiltration using a MPS 44 membrane with a 250 D cutoff. The A-40926 concentrated solution obtained (58.6 L; 3.89 g/L) wasadjusted at pH 6.4 and stored at 4° C. until used.

Column Preparation and Purification.

XAD-7 HP resin (8 L) was suspended in a 1:1 water/methanol solution,filtered, and loaded into a proper glass-column (internal diameter 12cm) with a peristaltic pump.

The resin was then washed with water and equilibrated with 6 BV of asodium carbonate aqueous solution buffered at pH 6 prepared bydissolving 5 g of sodium carbonate per liter of water and adjusting thepH with acetic acid.

A portion of concentrated broth containing 194 g of A-40926 was loadedinto the XAD-7 HP column. The resin was then washed with the followingtwo buffered solutions, at a flow rate of ½ BV/hour, in order toeliminate part of the hydrophilic and the colored substances present: 3BV (24 L) of aq. 0.5% Acetic Acid solution adjusted at pH 5 with 30%sodium hydroxide; 5 BV (40 L) of a 8:2 mixture water/acetone with 5 mLof acetic acid/L of water.

A-40926 was finally eluted with 8 BV (64 L) of a 1:1 water/acetonemixture acidified with 5 mL of acetic acid/L of water. 16 fractions of 4L each were collected. The rich fractions (from 5 to 15) in whichA-40926 concentration was greater than 0.5 g/L were gathered togetherobtaining a solution containing 163.4 g of A-40926 (43 L, 3.8 g/L). Thecolumn yield was 81.3%. The other fractions (200 L) containing 0.23 g/L(45.3 g; 22.2%) of less pure A-40926 were discharged.

After the elution the resin was regenerated with 6 BV (55 L) of NaOH0.5%/isopropanol (1:1) mixture, and finally washed to neutral with 10 BVof water.

Charcoal Treatment.

The collected fractions were adjusted at pH 2.5 with HCl 37% (70 mL) andthen decolorized with 50 g charcoal type PA 200 (0.3 g/g of A-40926).The suspension obtained was stirred for 2 hours at room temperature andthen filtered through a KS 50 filter (d=25 cm, time=2.5 hours),obtaining 45.6 L of a slightly yellow A-40926 solution (3.5 g/L;yield=96.4%).

Concentration.

The decolorized solution was adjusted at pH 7 with NaOH 30% (230 mL) andconcentrated by nanofiltration and ultrafiltration. The use of thesetechniques was important for the elimination of the hydrophilicsubstances that were detected on the HPLC chromatograms at Rt=2-4minutes. When the retentate was concentrated to 1/10 of the startingvolume (4 L), the same volume of water was added and the solutionobtained was concentrated again. This concentration/dilution step wasrepeated three times in order to reduce the residual acetone to 0.25%.The final solution (2.2 L, 146.3 g of A-40926, 66.5 g/L, yield=91.5%)was analyzed by HPLC. The purification yield was 75.4%.

A-40926 Crystallization.

A 300 mL portion of the A-40926 solution (19.9 g of A-40926) was furtherconcentrated to 100 mL by using a laboratory scale ultrafilter and thenheated at 60-65° C. The pH of this solution was adjusted at 7 (30%NaOH), and 1.2 mL of 5:1 acetone/isopropanol mixture per mL ofconcentrated solution was added drop wise at this temperature. Theresulted mixture was left to cool at 20° C. After 1.5 hours, the solidobtained was filtered, washed on the filter with acetone, and dried at40° C. for 15 hours. 20.6 g of product (HPLC assay 82.0%; A-40926 16.9g) was obtained. The precipitation yield was 84.9%. The overall yield,starting from the filtered broth, was about 64%.

A-40926 Preparation on CG-71

Column Preparation

CG-71 resin (350 mL) was poured into a glass-column (internal diameter=4cm) and washed with water. The resin was equilibrated with 3 BV of asodium carbonate solution, prepared by dissolving 5 g of sodiumcarbonate in water at pH 6 with acetic acid. 250 mL fermentation broth(pH 7) containing 14.7 g of A-40926 was loaded into the column (42 g/Lresin). The resin was washed with the following three solutions: 1050 mL(3 BV) of aqueous solution of sodium carbonate (5 g/L) adjusted at pH 6with acetic acid; 1750 mL (5 BV) of aqueous solution of sodium carbonate(5 g/L) adjusted at pH 8 with acetic acid; 3150 mL (9 BV) of aqueoussolution of sodium carbonate (5 g/L) adjusted at pH 9 with acetic acid.

The activity was then eluted with 10 BV of demineralized water. 20fractions of 500 mL each were collected. Fractions 12 to 15 were pooledtogether, obtaining 2.2 L purified solution containing 11.7 g of A-40926(yield=79.6%). This solution was then concentrated by ultrafiltrationand the concentrate solution was further diluted with demineralizedwater and ultrafiltered again. The solution obtained was furtherconcentrated under reduced pressure to 50 mL.

A-40926 Crystallization.

The concentrated solution was heated at 60° C. and treated understirring with a 5:1 acetone/IPA mixture (60 mL). The mixture was thenslowly cooled at room temperature. The solid obtained was filtered,washed with acetone on the filter, and dried under vacuum at 35° C. for80 hours. 8.9 g of purified A-40926 (HPLC assay 84.2%) was obtained. Theoverall yield was 51%.

Alternative Amidation Step in Dalbavancin Synthesis UsingN-Methyl-2-Pyrrolidine (NMP) as a Solvent

MA mixture was added portion wise under stirring to a 1:1 NMP/MeOHmixture (64 mL). Stirring at 20-25° C. was continued until completesolution, then DMEPA (2.42 mL; 1.96 mol/eq_(MA)) and HOBT (1.06 g; 0.71mol/eq_(MA)) were added. The pH of the reaction mixture (checked on asample diluted 1:10 with water) was adjusted to 3.0 with 9.37 mL of 15%HCl in NMP (previously prepared from 34.0 mL HCl 37% dissolved in 57.7mL NMP). Then a solution of DCC (3.17 g; 1.57 mol/eq_(MA)) in NMP/MeOH1:1 (12.7 mL) was added under stirring. The reaction was monitored byHPLC. The reaction was complete after about 6 hours (MA-A-1 88.9%, MA7.3%, ISO 3.7%). This experiment suggests that NMP can be a convenientalternative to DMSO for the amidation reaction. The whole process wasnot influenced by this solvent change and the final Dalbavancin obtainedwas chemically equivalent to other batches.

Alternative Method of Dalbavancin Preparation: One-Pot Procedure

10 g of A-40926 complex (HPLC titer 80.66%, 4.6 mmole) was suspended in24 mL of MeOH under stirring at room temperature in a 100 mL glassreactor. The mixture was cooled at 0° C., and a solution of 4 g of HCl(g) in 16.4 mL of MeOH was added to complete the product solubilization.The temperature was then left to rise to 20° C. while stirring wascontinued for additional 24 hours.

After this time, 40 mL of DMSO and 0.4 g of HOBT were added to thereaction mixture.

1,1-dimethyamine propylamine was then added, adjusting the pH of theresulting reaction mixture between 3-3.1 (measured after diluting asample 9:1 with water). 1.8 g of solid DCC was then added and stirringwas continued for additional 15 hours. After this time the reactionmixture was transferred in a 1 L glass reactor and diluted with 80 mL ofwater. The pH was then brought to 12 by adding 240 mL of 15% NaOH.Stirring was continued for additional 60 minutes, and the mixture wasacidified at pH 2.8 with 260 mL of 15% aq. HCl. About 800 mL of finalclear solution containing 6.4 g of Dalbavancin was obtained (yield=76%).

HPLC analyses showed that the profile of the product obtained iscomparable with that obtained with the other manufacturing processes.

N¹⁵,N¹⁵-Dialkyl Antibiotic Compounds

The present invention provides N¹⁵,N¹⁵-dialkyl antibiotic compoundsuseful, for example, for preventing and/or treating microbial infectionsin mammals. For convenience, in the description herein, dalbavancincompounds are numbered according to U.S. Pat. No. 5,750,509 and asdepicted in FIG. 26.

In certain embodiments, the present invention provides N¹⁵,N¹⁵-dialkylantibiotic compounds according to formula (III), or a pharmaceuticallyacceptable salt or solvate thereof:

In certain embodiments, the present invention provides N¹⁵,N¹⁵-dialkylantibiotic compounds according to formula (IV), or a pharmaceuticallyacceptable salt or solvate thereof:

Formulas (III) and (IV) provide the peptide core of the N¹⁵,N¹⁵-dialkylantibiotic compounds of the invention. The peptide comprises seven aminoacids with the amino terminus at the right side, as depicted in formulas(III) and (IV), and the carboxy terminus at the left. According to thisaspect of the invention, the N¹⁵,N¹⁵-dialkyl antibiotic compoundscomprise two alkyl substituents on the amino terminal nitrogen, or N¹⁵.In other words, in formulas (III) and (IV) R′ and R″ are alkyl.

In preferred embodiments, R′ and R″ are C₁₋₄ alkyl. In certainembodiments, R¹ and R^(1′) are each independently selected from propyl,ethyl and methyl. In further embodiments, R¹ and R^(1′) are eachindependently selected from ethyl and methyl. In more preferredembodiments, at least one of R¹ and R^(1′) is methyl. In most preferredembodiments, R¹ and R^(1′) are methyl.

The remainder of the molecule can be modified in the same manner asdalbavancin compounds known to those of skill in the art. Accordingly, Xis an aminoalkylamino group, as defined in the sections above. Exemplaryaminoalkylamino groups are described in U.S. Pat. No. 5,750,509. Incertain embodiments, X is N,N-dimethylaminopropylamino.

Thus, in particular embodiments, the invention provides compoundsaccording to formula (V), or a pharmaceutically acceptable salt orsolvate thereof:

The N¹⁵,N¹⁵-dialkyl antibiotic compounds of the invention can beglycosylated in one or more positions, as is known to those of skill inthe art. In preferred embodiments, the N¹⁵,N¹⁵-dialkyl antibioticcompounds are glycosylated at positions G and M of formula (III) or(IV). In particular embodiments, M is hydrogen or a sugar moiety. Forexample, M can be hydrogen, α-D-mannopyrannosyl or6-O-acetyl-α-D-mannopyrannosyl. In particular embodiments, G is hydrogenor a sugar moiety. For example, G can be hydrogen or glucuronamine.

In certain embodiments, one or both of the sugar moieties can beacylated or acetylated, or both. For example, when G is glucuronamine,the glucuronamine moiety can be acylated with a fatty acid. The fattyacid can be any fatty acid known to those of skill in the art. Inparticular embodiments, the fatty acid is selected from the groupconsisting of 8-methyl nonanoic acid, n-decanoic acid, 9-methyl-decanoicacid, n-undecanoic acid, 10-methyl-undecanoic acid, n-dodecanoic acid,11-methyl-dodecanoic acid, n-tridecanoic acid, 12-methyl-tridecanoicacid and n-tetradecanoic acid. In preferred embodiments, the fatty acidis a C₁₂ fatty acid. In particular embodiments, the fatty acid is10-methyl-undecanoic acid.

In certain embodiments, the present invention provides a compoundaccording to formula (VI), or a pharmaceutically acceptable salt orsolvate thereof:

In formula (VI), R² is C₁₀₋₁₄ acyl. In particular embodiments, R² isselected from the group consisting of 8-methyl nonanoic acid, n-decanoicacid, 9-methyl-decanoic acid, n-undecanoic acid, 10-methyl-undecanoicacid, n-dodecanoic acid, 111-methyl-dodecanoic acid, n-tridecanoic acid,12-methyl-tridecanoic acid and n-tetradecanoic acid. In preferredembodiments, R² is a C₁₂ fatty acid. In particular embodiments, R² is10-methyl-undecanoic acid.

As described in the synthetic sections below, N¹⁵,N¹⁵-dimethylantibiotic compounds can be synthesized from dalbavancin compounds.Accordingly, certain N¹⁵,N¹⁵-dimethyl antibiotic compounds of theinvention possess the sugar moieties and fatty acids of dalbavancin A₀,A₁, B₀, B₁, C₀ and C₁.

In particular embodiments, the present invention providesN¹⁵,N¹⁵-dimethyl antibiotic compounds. An N¹⁵,N¹⁵-dimethyl antibioticcompound comprises formula (I) or (II) with two methyl groups atN¹⁵,N¹⁵N¹⁵-dimethyl antibiotic compound can be glycosylated with one ormore sugar moieties as described above. In certain embodiments, aN¹⁵,N¹⁵-dimethyl antibiotic compound is acylated on one or more of thesesugar moieties. In preferred embodiments, the acyl group is10-methyl-undecanoic acid.

In preferred embodiments, the present invention provides theN¹⁵,N¹⁵-dimethyl antibiotic compound according to the followingstructure (VII), or a pharmaceutically acceptable salt or solvatethereof:

In certain embodiments, the N¹⁵,N¹⁵-dialkyl antibiotic compound of theinvention is purified. As used herein, the term purified indicates thatthe N¹⁵,N¹⁵-dialkyl antibiotic compound is enriched relative to otherdalbavancin compounds in a composition. For example, the N¹⁵,N¹⁵-dialkylantibiotic compound can be enriched relative to a mixture in which theN¹⁵,N¹⁵-dialkyl antibiotic compound is produced, for instance a mixturederived from a fermentation broth as described in the examples below. Incertain embodiments, the N¹⁵,N¹⁵-dialkyl antibiotic compound is enrichedtwo, three, five, ten, one hundred, one thousand or ten thousand fold.

In further embodiments, the N¹⁵,N¹⁵-dialkyl antibiotic compound isisolated. As used herein, the term isolated indicates that theN¹⁵,N¹⁵-dialkyl antibiotic compound is enriched relative tonon-dalbavancin compounds in a composition. For example, theN¹⁵,N¹⁵-dialkyl antibiotic compound can be enriched relative to amixture in which the N¹⁵,N¹⁵-dialkyl antibiotic compound is produced,for instance a mixture derived from a fermentation broth as described inthe examples below. In certain embodiments, the N¹⁵,N¹⁵-dialkylantibiotic compound is enriched two, three, five, ten, one hundred, onethousand or ten thousand fold.

In still further embodiments, the N¹⁵,N¹⁵-dialkyl antibiotic compound ispurified and isolated. As used herein, the term purified and isolatedindicates that the N¹⁵,N¹⁵-dialkyl antibiotic compound is enrichedrelative to non-dalbavancin compounds and relative to other dalbavancincompounds in a composition. For example, the N¹⁵,N¹⁵-dialkyl antibioticcompound can be enriched relative to a mixture in which theN¹⁵,N¹⁵-dialkyl antibiotic compound is produced, for instance a mixturederived from a fermentation broth as described in the examples below. Incertain embodiments, the N¹⁵,N¹⁵-dialkyl antibiotic compound is enrichedtwo, three, five, ten, one hundred, one thousand or ten thousand fold.

N¹⁵,N¹⁵-Dialkyl Antibiotic Compounds

The present invention also provides N¹⁵,N¹⁵-dialkyl antibiotic compoundshaving a carboxylic acid group at carbonyl 63. These compounds areuseful, for example, for preparing N¹⁵,N¹⁵-dialkyl antibiotic compoundsof the invention. In certain embodiments, these N¹⁵,N¹⁵-dialkylantibiotic compounds are also useful for preventing and/or treatingmicrobial infections in mammals.

In certain embodiments, the present invention provides N¹⁵,N¹⁵-dialkylantibiotic compounds according to formula (VIII), or a pharmaceuticallyacceptable salt or solvate thereof:

In certain embodiments, the present invention provides N¹⁵,N¹⁵-dialkylantibiotic compounds according to formula (IX), or a pharmaceuticallyacceptable salt or solvate thereof:

Formulas (VIII) and (IX) provide the peptide core of the N¹⁵,N¹⁵-dialkylantibiotic compounds of this aspect of the invention. The peptidecomprises seven amino acids with the amino terminus at the right side,as depicted in formulas (III) and (IV), and the carboxy terminus at theleft. According to this aspect of the invention, the N¹⁵,N¹⁵-dialkylantibiotic compounds comprise two alkyl substituents on the aminoterminal nitrogen, or N¹⁵. In other words, in formulas (I) and (II) R¹and R^(1′) are alkyl. Further, in Formulas (VIII) and (IX), X is OH asin antibiotic A 40926 compounds known to those of skill in the art.

In preferred embodiments, R¹ and R^(1′) are C₁₋₄ alkyl. In certainembodiments, R¹ and R^(1′) are each independently selected from propyl,ethyl and methyl. In further embodiments, R¹ and R^(1′) are eachindependently selected from ethyl and methyl. In more preferredembodiments, at least one of R¹ and R^(1′) is methyl. In most preferredembodiments, R¹ and R^(1′) are methyl.

The N¹⁵,N¹⁵-dialkyl antibiotic compounds of the invention can beglycosylated in one or more positions, as is known to those of skill inthe art. In preferred embodiments, the N¹⁵,N¹⁵-dialkyl antibioticcompounds are glycosylated at positions G and M of formulas (VIII) and(IX). In particular embodiments, M is hydrogen or a sugar moiety. Forexample, M can be hydrogen, α-D-mannopyrannosyl or6-O-acetyl-α-D-mannopyrannosyl. In particular embodiments, G is hydrogenor a sugar moiety. For example, G can be hydrogen or glucuronamine.

In certain embodiments, one or both of the sugar moieties can beacylated or acetylated, or both. For example, when G is glucuronamine,the glucuronamine moiety can be acylated with a fatty acid. The fattyacid can be any fatty acid known to those of skill in the art. Inparticular embodiments, the fatty acid is selected from the groupconsisting of 8-methyl nonanoic acid, n-decanoic acid, 9-methyl-decanoicacid, n-undecanoic acid, 10-methyl-undecanoic acid, n-dodecanoic acid,11-methyl-dodecanoic acid, n-tridecanoic acid, 12-methyl-tridecanoicacid and n-tetradecanoic acid. In preferred embodiments, the fatty acidis a C₁₂ fatty acid. In particular embodiments, the fatty acid is10-methyl-undecanoic acid.

In certain embodiments, the present invention provides a compoundaccording to formula (X), or a pharmaceutically acceptable salt orsolvate thereof:

In formula (X), R² is C₁₀₋₁₄ acyl. In particular embodiments, R² isselected from the group consisting of 8-methyl nonanoic acid, n-decanoicacid, 9-methyl-decanoic acid, n-undecanoic acid, 10-methyl-undecanoicacid, n-dodecanoic acid, 11-methyl-dodecanoic acid, n-tridecanoic acid,12-methyl-tridecanoic acid, and n-tetradecanoic acid. In preferredembodiments, R² is a C₁₂ fatty acid. In particular embodiments, R² is10-methyl-undecanoic acid.

As described in the synthetic sections below, the N¹⁵,N¹⁵-dimethylantibiotic compounds can be synthesized from antibiotic A 40926compounds. Accordingly, certain N¹⁵,N¹⁵-dimethyl antibiotic compounds ofthe invention possess the sugar moieties and fatty acids of antibiotic A40926 factor A₀, A₁, B₀, B₁, C₀ and C₁.

In particular embodiments, the present invention providesN¹⁵,N¹⁵-dimethyl antibiotic compounds. An N¹⁵,N¹⁵-dimethyl antibioticcompound comprises formula (VIII) or (IX) with two methyl groups at N¹⁵.An N¹⁵,N¹⁵-dimethyl antibiotic compound can be glycosylated with one ormore sugar moieties as described above. In certain embodiments, aN¹⁵,N¹⁵-dimethyl antibiotic compound is acylated on one or more of thesesugar moieties. In preferred embodiments, the acyl group is IO-methyl-undecanoic acid.

In preferred embodiments, the present invention provides theN¹⁵,N¹⁵-dimethyl antibiotic compound according to the followingstructure (XI), or a pharmaceutically acceptable salt or solvatethereof:

In certain embodiments, the N¹⁵,N¹⁵-dialkyl antibiotic compound of theinvention is purified. As used herein, the term purified indicates thatthe N¹⁵,N¹⁵-dialkyl antibiotic compound is enriched relative to otherantibiotic compounds in a composition. For example, the N¹⁵,N¹⁵-dialkylantibiotic compound can be enriched relative to a mixture in which theN¹⁵,N¹⁵-dialkyl antibiotic compound is produced, for instance a mixturederived from a fermentation broth as described in the examples below. Incertain embodiments, the N¹⁵,N¹⁵-dialkyl antibiotic compound is enrichedtwo, three, five, ten, one hundred, one thousand or ten thousand fold.

In further embodiments, the N¹⁵,N¹⁵-dialkyl antibiotic compound isisolated. As used herein, the term isolated indicates that theN¹⁵,N¹⁵-dialkyl antibiotic compound is enriched relative tonon-antibiotic compounds in a composition. For example, theN¹⁵,N¹⁵-dialkyl antibiotic compound can be enriched relative to amixture in which the N¹⁵,N¹⁵-dialkyl antibiotic compound is produced,for instance a mixture derived from a fermentation broth as described inthe examples below. In certain embodiments, the N¹⁵,N¹⁵-dialkylantibiotic compound is enriched two, three, five, ten, one hundred, onethousand or ten thousand fold.

In still further embodiments, the N¹⁵,N¹⁵-dialkyl antibiotic compound ispurified and isolated. As used herein, the term purified and isolatedindicates that the N¹⁵,N¹⁵-dialkyl antibiotic compound is enrichedrelative to non-antibiotic compounds and relative to other dalbavancincompounds in a composition. For example, the N¹⁵,N¹⁵-dialkyl antibioticcompound can be enriched relative to a mixture in which theN¹⁵,N¹⁵-dialkyl antibiotic compound is produced, for instance a mixturederived from a fermentation broth as described in the examples below. Incertain embodiments, the N¹⁵,N¹⁵-dialkyl antibiotic compound is enrichedtwo, three, five, ten, one hundred, one thousand or ten thousand fold.

Methods of Making the Compounds of the Invention

The compounds of the invention can be made according to any methodapparent to those of skill in the art for preparing such N¹⁵,N¹⁵-dialkylantibiotic compounds.

For instance, in certain embodiments, an N¹⁵,N¹⁵-dialkyl antibioticcompound or composition of the invention can be prepared by alkylatingthe corresponding N¹⁵-monomethyl compound or composition by anytechnique apparent to one of skill in the art. For example, anN¹⁵-monomethyl compound or composition can be methylated by contactingthe compound or composition with a mixture of HCHO, NaBH₃CN, DMF, H₂Oand NaHCO₃ at room temperature as illustrated in Scheme 1. In aparticular embodiment, an N¹⁵-monomethyl dalbavancin compound orcomposition is dissolved in water and DMF, to which formaldehyde andsodium bicarbonate are added followed by NaBH₃CN to yield theN¹⁵,N¹⁵-dimethyl antibiotic compound or composition.

The corresponding N¹⁵-monomethyl compound or composition can be preparedaccording to published techniques, such as those described extensivelyin U.S. Pat. No. 5,750,509 and U.S. Patent Application Publication No.US 2004/0142883, the contents of which are incorporated by reference intheir entireties. To the extent that the compounds or compositions haveprotecting groups on their N¹⁵ nitrogen, they can be removed by anytechnique known to the person of skill in the art.

Suitable starting materials include dalbavancin A₀, A₁, B₀, B₁, C₀ andC₁ and antibiotic A 40926 factors A₀, A₁, B₀, B₁, C₀ and C₁. As shown inthe examples below, N¹⁵,N¹⁵-dimethyl dalbavancin B₀ was prepared fromdalbavancin B₀ according to this method. The preparation of thesedalbavancin compounds is described extensively in U.S. PatentApplication Publication No. US 2004/0142883. These dalbavancin compoundshave the following structures:

wherein R is as follows: Dalbavancin Compound R A₀ —CH(CH₃)₂ A₁—CH₂CH₂CH₃ B₀ —CH₂CH(CH₃)₂ B₁ —CH₂CH₂CH₂CH₃ C₀ —CH₂CH₂CH(CH₃)₂ C₁—CH₂CH₂CH₂CH₂CH₃

In further embodiments, an N¹⁵,N¹⁵-dialkyl antibiotic compound of theinvention can be prepared from antibiotic A 40926 fermentation broth.The fermentation broth comprising antibiotic A 40926 and relatedcompounds can be prepared according to techniques described, forexample, in U.S. Pat. No. 5,750,509 and U.S. Patent ApplicationPublication No. U.S. 2004/0142883. The broth can be purified to yieldthe desired antibiotic A 40926 compounds, which can be esterified,amidated and hydrolyzed according to U.S. Pat. No. 5,750,509 and U.S.Patent Application Publication No. US 2004/0142883 to yield a mixture ofdalbavancin compounds. N¹⁵,N¹⁵-dialkyl antibiotic compounds can bepurified and/or isolated from the mixture according to any techniqueapparent to those of skill in the art. In the examples below, HPLCtechniques are used to purify N¹⁵,N¹⁵-dialkyl antibiotic compounds ofthe invention.

Compositions

In another aspect, the present invention provides compositionscomprising one or more compounds of the invention. Generally, thecompositions of the invention comprise a compound of the invention andone or more other compounds. The other compounds can be compounds of theinvention, compounds known to those of skill in the art, compounds yetto be discovered or published or other compounds.

In certain embodiments, the compositions are pharmaceuticalcompositions, described in more detail in the sections below.

In particular embodiments, the compositions comprise a N¹⁵,N¹⁵-dialkylantibiotic compound of the invention and a dalbavancin or antibiotic A40926 compound. The dalbavancin or antibiotic A 40926 compound can beany dalbavancin or antibiotic A 40926 compound known to those of skillin the art. Exemplary dalbavancin compounds include those described inU.S. Pat. No. 5,750,509 and U.S. Patent Application Publication No. US2004/0142883, the contents of which are hereby incorporated by referencein their entireties. Exemplary antibiotic A 40926 compounds aredescribed in U.S. Pat. Nos. 4,935,238, 4,868,171 and 4,782,042, thecontents of which are hereby incorporated by reference in theirentireties.

Exemplary dalbavancin compounds include dalbavancin A₀, A₁, B₀, B₁, C₀and C₁. The preparation of these dalbavancin compounds is describedextensively in U.S. Patent Application Publication No. US 2004/0142883.These dalbavancin compounds are further described in the sections above.Of course, the compositions of the invention can comprise additionaldalbavancin compounds not listed among the exemplary dalbavancincompounds above.

In certain embodiments the compositions comprise multimers of compoundsof the invention. The multimers can be dimers, trimers or larger. Themultimers can be homomultimers, heteromultimers or a mixture ofhomomultimers and heteromultimers. For instance, the multimer cancomprise a combination of any of the factors present in the dalbavancincomposition, including any of dalbavancin factors A₀, A₁, B₀, B₁, B₂,C₀, C₁, D₀, D₁, MAG, or isoB₀. For example, the multimer may comprise aN¹⁵-alkyl dalbavancin compound and a N¹⁵,N¹⁵-dialkyl dalbavancincompound. In certain embodiments, the present invention also provideshomodimers of the compounds of the invention. In further embodiments,the present invention provides heterodimers of the compounds of theinvention with dalbavancin compounds. The dalbavancin compounds can bedalbavancins of the invention or dalbavancins known to those of skill inthe art.

In certain embodiments, the compositions of the invention comprise asignificant amount of an N¹⁵,N¹⁵-dialkyl antibiotic compound of theinvention relative to dalbavancin or antibiotic A 40926 compounds. Incertain embodiments, relative to the dalbavancin or antibiotic A 40926compounds, the N¹⁵,N¹⁵-dialkyl antibiotic compound of the invention isat least 0.05, 0.1, 0.15, 0.2, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.6,1.7, 1.75, 1.8, 1.9, 2.0, 2.5, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70,80, 90, 95, 96, 97, 98 or 99% of the composition. In preferredembodiments, the compound is according to formula (V).

In certain embodiments, the compositions of the invention comprise asignificant amount of an N¹⁵,N¹⁵-dialkyl antibiotic compound of theinvention relative to dalbavancin compounds. In certain embodiments,relative to the dalbavancin compounds, the N¹⁵,N¹⁵-dialkyl antibioticcompound of the invention is at least 0.05, 0.1, 0.15, 0.2, 0.25, 0.5,0.75, 1.0, 1.25, 1.5, 1.6, 1.7, 1.75, 1.8, 1.9, 2.0, 2.5, 5, 10, 15, 20,25, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98 or 99% of thecomposition. In preferred embodiments, the compound is according toformula (V).

In certain embodiments, the compositions of the invention comprise asignificant amount of an N¹⁵,N¹⁵-dialkyl antibiotic compound of theinvention relative to antibiotic A 40926 compounds. In certainembodiments, relative to the antibiotic A 40926 compounds, theN¹⁵,N¹⁵-dialkyl antibiotic compound of the invention is at least 0.05,0.1, 0.15, 0.2, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.6, 1.7, 1.75, 1.8,1.9, 2.0, 2.5, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 96,97, 98 or 99% of the composition. In preferred embodiments, the compoundis according to formula (V).

In certain embodiments, the compositions of the invention comprise asignificant amount of an N¹⁵,N¹⁵-dialkyl antibiotic compound of theinvention relative to other compounds. In certain embodiments, theN¹⁵,N¹⁵-dialkyl antibiotic compound of the invention is at least 0.05,0.1, 0.15, 0.2, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.6, 1.7, 1.75, 1.8,1.9, 2.0, 2.5, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 96,97, 98 or 99% of the composition relative to other compounds. Inpreferred embodiments, the compound is according to formula (V).

In further embodiments, the present invention provides compositionscomprising dalbavancin A₀, A₁, B₀, B₁, C₀ and C₁ together with anN¹⁵,N¹⁵-dialkyl antibiotic compound of the invention wherein the amountof the compound of the invention is enriched relative to one or more orall of dalbavancin A₀, A₁, B₀, B₁, C₀ and C₁. The composition can beenriched, for example, so that the N¹⁵,N¹⁵-dialkyl antibiotic compoundis at least 0.05, 0.1, 0.15, 0.2, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.6,1.7, 1.75, 1.8, 1.9, 2.0, 2.5, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70,80, 90, 95, 96, 97, 98 or 99% of the composition relative to one or moreor all of dalbavancin A₀, A₁, B₀, B₁, C₀ and C₁ In preferredembodiments, the compound is according to formula (V).

In certain embodiments, the present invention provides compositionscomprising dalbavancin B₀ and an N¹⁵,N¹⁵-dialkyl antibiotic compound ofthe invention. In further embodiments, the present invention providescompositions comprising dalbavancin B₁ and an N¹⁵,N¹⁵-dialkyl antibioticcompound of the invention. In further embodiments, the present inventionprovides compositions comprising dalbavancin B₀, dalbavancin B₁ and anN¹⁵,N¹⁵-dialkyl antibiotic compound of the invention. In preferredembodiments, the N¹⁵,N¹⁵-dialkyl antibiotic compound of the invention isaccording to formula (V). In particularly preferred embodiments, thepresent invention provides compositions comprising the compound offormula (V), dalbavancin B₀ and dalbavancin B₁. These compositions canbe purified, isolated or purified and isolated. In certain embodiments,the compositions are 90, 91, 92, 93, 94, 95, 96, 97, 98, 98.5, 99 or99.5% pure. By “pure” is meant that the compositions comprise thatamount of the compounds of the composition. For instance, thecomposition comprising the compound of formula (V), dalbavancin B₀ anddalbavancin B₁ would comprise at least 90, 91, 92, 93, 94, 95, 96, 97,98, 98.5, 99 or 99.5% of the three compounds relative to other compoundsin the composition. Purity can be assessed by any means known to thoseof skill in the art such as HPLC, for instance by % area under thecurve.

In the compositions of the invention, the amount of each component canbe calculated by weight, by molar amount or by any other technique knownto those of skill in the art.

Methods of Use

Methods are provided for administration of a N¹⁵,N¹⁵-dialkyl antibioticcompound or composition of the invention to an individual in need oftreatment for a bacterial infection. In one embodiment, theN¹⁵,N¹⁵-dialkyl antibiotic compound is according to formula (V).Treatment can include prophylaxis, therapy, or cure. Methods includeadministration of a N¹⁵,N¹⁵-dialkyl antibiotic compound or compositionin a therapeutically or prophylactically effective amount.

As used herein, “therapeutically effective amount” refers to the amountof a compound or composition that, when administered to a subject fortreating a disease, will render a desired therapeutic outcome (e.g.,reduction or elimination of a bacterial infection). A “therapeuticallyeffective amount” can vary depending on, inter alia, the compound, thedisease and its severity, and the age, weight, etc., of the subject tobe treated, and may be administered in one or more doses. A“prophylactically effective amount” refers to an amount of aN¹⁵,N¹⁵-dialkyl antibiotic compound or composition sufficient to preventor reduce severity of a future bacterial infection when administered toan individual who is susceptible to and/or who may contract a bacterialinfection, e.g., by virtue of a medical procedure or stay in thehospital, or exposure to an individual with a bacterial infection. AN¹⁵,N¹⁵-dialkyl antibiotic compound or composition can be administeredin a pharmaceutically acceptable carrier.

A N¹⁵,N¹⁵-dialkyl antibiotic compound or composition can be administeredas a “unit dose” in a N¹⁵,N¹⁵-dialkyl antibiotic formulation whichincludes an amount of the compound sufficient to provide atherapeutically or prophylactically effective plasma level of thecompound for several days, at least about 5 days, one week, or 10 days,when administered to an individual. In some embodiments, the compound isaccording to formula (V). In some embodiments, the compound is acomponent of a formulation, which includes an amount of the compoundsufficient to provide therapeutically or prophylactically effectivelevel of the compound for several days, often at least about 5 days, oneweek, or 10 days. The dosing interval, or time between doses, can be,for example, any of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more days.

As used herein, the terms “individual” or “subject” or “patient” areused interchangeably and refer to a vertebrate, typically a mammal,often a human.

In addition, a compound or composition of the invention can beformulated with a stabilizer that inhibits degradation of the compound,the composition, and/or one or more other N¹⁵,N¹⁵-dialkyl antibiotic orother antibiotic components present in the composition. In someembodiments, the stabilizer is selected from the group consisting ofmannitol, lactose, sucrose, sorbitol, glycerol, cellulose, trehalose,maltose, raffinose, and mixtures thereof. In one embodiment, aformulation comprises mannitol. In another embodiment, a formulationfurther comprises lactose. In one embodiment, the composition can beformulated with a 1:2 weight ratio of mannitol: N¹⁵,N¹⁵-dialkylantibiotic compound. In another embodiment, the composition can beformulated with a 1:1:4 weight ratio of mannitol:lactose:N¹⁵,N¹⁵-dialkyl antibiotic compound.

In some embodiments, a composition or other formulation comprises aN¹⁵,N¹⁵-dimethyl antibiotic compound, a dalbavancin compound, or acombination thereof. A composition or other formulation can beadministered at a dosage that results in therapeutically effective(i.e., bactericidal) plasma levels of the drug for several days, oftenat least about 5 to about 10 days, often at least about one week. Forexample, N¹⁵,N¹⁵-dialkyl antibiotic compounds or compositions can bemaintained in plasma at or above the minimum bactericidal concentrationof about 4 mg/l for at least 5 days. N¹⁵,N¹⁵-dialkyl antibioticcompounds or compositions can be maintained at a plasma level of atleast about 5 mg/l, at least about 10 mg/l, at least about 20 mg/l, atleast about 30 mg/l, at least about 40 mg/l, for at least 5 days, atleast about one week or longer. Plasma levels of N¹⁵,N¹⁵-dialkylantibiotic compounds or compositions can be measured by methods that arewell known in the art, such as liquid chromatography, mass spectrometry,or microbiological bioassay.

Upper limits for N¹⁵,N¹⁵-dialkyl antibiotic compound or compositionplasma concentration levels can be dictated by dosages that inhibitunacceptable adverse effects in the patient population treated.

A N¹⁵,N¹⁵-dialkyl antibiotic compound or composition can be administeredin a single dose or in multiple doses. When administered as a singledose, a N¹⁵,N¹⁵-dialkyl antibiotic compound or composition can beformulated to contain a sufficient amount of the N¹⁵,N¹⁵-dialkylantibiotic compound(s) to effect antibacterial properties in vivo for atleast 5 days, alternatively for at least 6 days, alternatively for atleast 7 days, alternatively for at least 8 days, alternatively for atleast 9 days, alternatively for at least 10 days, alternatively for atleast 11 days, alternatively for at least 12 days, alternatively for atleast 13 days, alternatively for at least 14 days, alternatively for atleast 15 days.

When multiple doses are employed, a N¹⁵,N¹⁵-dialkyl antibiotic compoundor composition can be administered weekly for two or more weeks. In oneembodiment, a N¹⁵,N¹⁵-dialkyl antibiotic compound or composition can beadministered in at least two doses, often in two doses about 5 to about10 days apart, more often once a week for two weeks. In certainembodiments, such a regimen provides significant advantages overconventional antibiotic treatment protocols.

A N¹⁵,N¹⁵-dialkyl antibiotic compound or composition can also beadministered in multiple doses two or more days or at least one weekapart, or in one or more biweekly doses. In some embodiments, aN¹⁵,N¹⁵-dialkyl antibiotic compound or composition can be administeredweekly, followed by biweekly, or monthly administration. In someembodiments, a N¹⁵,N¹⁵-dialkyl antibiotic compound or composition can beadministered at weekly intervals for 2, 3, 4, 5, 6, or more weeks.

Most advantageously, daily dosing of a N¹⁵,N¹⁵-dialkyl antibioticcompound or composition is not required because higher, less frequentdoses can be used. Single or multiple doses can range, for example, fromabout 0.1 to about 5 grams. A single dose of about 0.1 to about 4 grams,e.g., about 3 grams, can be administered for various infectiontreatments. Where multiple doses are administered, for example, weekly,each dose can range, for example, from about 0.25 to about 5 grams.

For embodiments in which a single dose can be administered to treat aninfection, the amount of the dose can be, for example, about 0.1 toabout 5 grams, or about 0.5 to about 4 grams, or about 1 to about 3.5grams, or about 2 to about 3 grams, e.g., about 3 grams. In someembodiments, a single dose of about 1, 1.5, 2, 2.5, or 3 grams can beadministered for treatment of a bacterial infection. For embodiments inwhich a single dose can be administered for prophylaxis, the amount ofthe dose may be, for example, about 0.1 to about 3 grams, or about 0.1to about 1 gram, e.g., about 0.5 or about 0.25 gram.

In dosing schemes that include multiple dosages, the individual dosagesmay be the same or different. In some embodiments, a first, higher dosecan be administered, that can be, for example, about 1.5 to 3 timeshigher, than one or more subsequent doses. For example, the first dosemay be about 0.5 grams to about 5 grams and the second dose about 0.25grams to about 2.5 grams, the first dose may be about 0.8 to about 2gram and the second dose about 0.4 to about 1 gram, or the first dosemay be about 0.4 to about 3 gram and the second dose about 0.2 to 1.5gram.

In some embodiments, at least two dosages are administered wherein thefirst dosage includes about twice as much of a N¹⁵,N¹⁵-dialkylantibiotic compound or composition as subsequent dosages. In anembodiment, a first dosage includes about 1 gram of the N¹⁵,N¹⁵-dialkylantibiotic compound or composition and a subsequent dosage of about 0.5gram. In another embodiment, a first dosage includes about 0.5 gram anda subsequent dosage of about 0.25 gram.

In some embodiments, a N¹⁵,N¹⁵-dialkyl antibiotic compound orcomposition can be administered in two doses of equal or differentamounts two or more days or at least about one week apart. For example,two doses of about 0.2 to about 1.5 grams of a N¹⁵,N¹⁵-dialkylantibiotic compound or composition can be administered about 5 to about10 days apart, or about 1 week apart. In one embodiment, a first dosageof about 1 gram and a second dosage of about 0.5 gram of a N¹⁵-dialkylantibiotic compound or composition can be administered about 1 weekapart.

In a multiple dosing regimen, the time between doses may range, forexample, from about 5 to about 10 days, often about one week. Dosefrequency can be, for example, two weekly doses, or multiple weeklydoses. The dosing interval, or time between doses, can be, for example,any of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, or more days. The number of doses given, canbe, for example, one, two, three, four, five, six or more doses, eachdose after the initial dose being given after the selected dosageinterval.

In the multiple dosing schemes of certain embodiments, the “troughlevel,” or the level of a N¹⁵,N¹⁵-dialkyl antibiotic compound orcomposition in plasma after a first dose, and just prior toadministration of a second dose, can be at least about 4 mg/l. Thetrough level of N¹⁵,N¹⁵-dialkyl antibiotic compound or composition atthe end of a dosing interval such as about one week can be at leastabout 20 mg/l, at least about 30 mg/l, or at least about 40 mg/l.

A N¹⁵,N¹⁵-dialkyl antibiotic compound or composition can be administeredparenterally, e.g., intramuscularly (i.m.), intravenously (i.v.),subcutaneously (s.c.), intraperitoneally (i.p.), or intrathecally(i.t.). The dosing schedule and actual dosage administered may varydepending on such factors as the nature and severity of the infection,the age, weight, and general health of the patient and the tolerance ofa particular patient to the N¹⁵,N¹⁵-dialkyl antibiotic compound orcomposition, but will be ascertainable to health professionals. In oneembodiment, a one gram intravenous dose of a N¹⁵,N¹⁵-dialkyl antibioticcompound or composition can be followed by a 0.5 gram intravenous doseone week later.

Administration and delivery of the drug to the patient, e.g.,intravenously, can be done at a controlled rate, so that theconcentration in the blood does not increase too quickly or causeprecipitation to occur. In some embodiments, a N¹⁵,N¹⁵-dialkylantibiotic compound or composition can be administered at an appropriaterate such that the drug forms a complex with endogenous protein(s) inthe bloodstream. Without intending to be bound to a particular theory,it can be believed that endogenous protein, such as human serum albumin,can form a complex in vivo with one or two molecules of aN¹⁵,N¹⁵-dialkyl antibiotic compound. When a sufficient amount ofN¹⁵,N¹⁵-dialkyl antibiotic composition is present, up to two moleculesof N¹⁵,N¹⁵-dialkyl antibiotic compound can bind to the endogenousprotein, and this complex can be formed by binding of separateN¹⁵,N¹⁵-dialkyl antibiotic compounds at two different binding sites.Alternatively, it is possible that dimeric N¹⁵,N¹⁵-dialkyl antibioticcan bind to a single binding site on the endogenous protein.

The infusion duration of the N¹⁵,N¹⁵-dialkyl antibiotic compound orcomposition can be, for example, about 1 minute to about 2 hours. Forexample, an infusion duration of about 30 minutes can be used with aN¹⁵,N¹⁵-dialkyl antibiotic compound or composition, and the dose can beabout 0.5 to about 1 gram. Intravenous administration under controlledrate conditions can generate concentrations of N¹⁵,N¹⁵-dialkylantibiotic compound or composition in the body that are in great excessof what can be achieved in the solution phase at physiological pH invitro. Although not wishing to be limited by theory, this may be due tothe formation of a complex of N¹⁵,N¹⁵-dialkyl antibiotic with endogenousprotein(s) such as serum albumin, which may increase the capacity ofplasma to absorb the N¹⁵,N¹⁵-dialkyl antibiotic compound or composition.

In certain embodiments, formation of a N¹⁵,N¹⁵-dialkyl antibioticcomplex in vitro or ex vivo can permit faster administration, such as atleast about 1 minute, at least about 10 minutes or at least about 20minutes. Such a complex can be achieved by mixing human serum albuminand/or another endogenous protein with a N¹⁵,N¹⁵-dialkyl antibioticcompound or composition, thereby forming the complex in vitro or exvivo, and then administering this complex to the treated patient.Alternatively, the human serum albumin or other endogenous protein maybe obtained from autologous sources or by expression from amicroorganism modified to contain the gene for the protein.

The amount of the N¹⁵,N¹⁵-dialkyl antibiotic compound or compositionadministered can be any of the dosages disclosed herein. The dose can bechosen such that one or more N¹⁵,N¹⁵-dialkyl antibiotic compounds willremain at a therapeutically or prophylactically effective (i.e.,bactericidal) plasma level for an extended period of time, such as atleast 5 days, or about one week or longer. Administration of a dose of aN¹⁵,N¹⁵-dialkyl antibiotic compound or composition which produces andmaintains bactericidal concentrations for at least about one week (orabout 5 to about 10 days) are preferred. A bactericidal concentrationcan be defined as the concentration of a N¹⁵,N¹⁵-dialkyl antibioticcompound or composition required to kill at least about 80%, at leastabout 85%, at least about 90%, or at least about any one of 95%, 96%,97%, 98% or 99% of the bacteria present at the initiation of an in vitroexperiment over a 24 hour period. A minimum bactericidal concentrationof a N¹⁵,N¹⁵-dialkyl antibiotic compound or composition in plasma can beabout 4 mg/l.

Examples of indications that can be prevented or treated using theN¹⁵,N¹⁵-dialkyl antibiotic compounds, compositions, and methods of theinvention include both complicated and uncomplicated SSTIs, blood streaminfections (BSI), catheter-related blood stream infections (CRBSI),osteomyelitis, prosthetic joint infections, surgical prophylaxis,endocarditis, hospital or community acquired pneumonia, pneumococcalpneumonia, empiric treatment of febrile neutropenia, joint spaceinfections, and device infections (e.g., pace makers and internalcardiac defibrillators). Gram-positive or antibiotic-resistant bacterialinfections can be prevented or treated, such as a Bacillus,Corynebacteria, Listeria, Enterococcus, Staphylococcus, Streptococcus,Neisseria, or Clostridium genus infection, in particular Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus hemolyticus,Streptococcus pyogenes, Streptococcus pneumoniae, Groups A and CStreptococcus, Enterococcus faecalis, Bacillus subtilis, Neisseriagonorrhoeae, or Clostridium difficile. Other infections that can beprevented or treated using the N¹⁵,N¹⁵-dialkyl antibiotic compounds,compositions, and methods of the invention include gram negativebacterial, such as a Bartonella, Brucella, Campylobacter, Enterobacter,Escherichia (as well as other Proteobacteria), Francisella,Helicobacter, Hemophilus, Klebsiella, Legionella, Leptospira,Morganella, Moraxella, Proteus, Providencia, Pseudomonas, Salmonella,Serratia, Shigella, Stenotrophomonas, Vibrio, and Yersinia genusinfection, in particular Escherichia coli, Proteus vulgaris, Pseudomonasaeruginosas, and yeast, such as Candida albicans, infections.

The invention also encompasses the prevention or treatment of otherinfectious bacteria using the compounds, compositions, and methods ofthe invention, such as Helicobacter pyloris, Borelia burgdorferi,Legionella pneumophilia, Mycobacteria sporozoites (sp.) (e.g. Mtuberculosis, M avium, M intracellulare, M kansaii, M gordonae),Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcus bovis,Streptococcus (anaerobic sps.), pathogenic Campylobacter sp.,Enterococcus sp., Haemophilus influenzae, Bacillus antracis,Corynebacterium diphtheriae, Corynebacterium sp., Erysipelothrixrhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacteraerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp.,Fusobacterium nucleatum, Streptobacillus moniliformis, Treponemapallidium, Treponema pertenue, Leptospira, and Actinomyces israelliinfection.

The prevention and treatment of infections and disorders describedherein may be accomplished using the compounds, compositions and methodsof the invention. In one embodiment, the N¹⁵,N¹⁵-dialkyl antibioticcompound is according to formula (V). In some embodiments, the subjecthas not previously been treated with one or more antibiotics, such asvancomycin, or teicoplanin. In other embodiments, the subject has notbeen previously treated with a N¹⁵,N¹⁵-dialkyl antibiotic compound orcomposition. In some embodiments, the patient has previously been testedfor bacterial resistance to an antibiotic. In other embodiments, thepatient has not been previously tested for bacterial resistance to anantibiotic.

The invention also encompasses methods for the prevention or treatmentof SSTIs. Patients who may benefit from this prevention or treatment mayhave either deep or superficial infections. SSTI may involve deeper softtissue and/or require significant surgical intervention, such as forexample a major abscess, infected ulcer, major burn, or deep andextensive cellulitis. Infected surgical wounds may also be prevented ortreated.

The clinical presentation of skin and skin structure infection may varyfrom mild folliculitis to severe necrotizing fasciitis. The mode ofacquisition may also vary with community-acquired skin and skinstructure infections, which are often preceded by injuries resultingfrom occupational exposure or recreational activities, and are usuallyassociated with a greater diversity of pathogens. Hospital-acquired skinand skin structure infections are generally associated with surgicalprocedures, the development of pressure sores, and catheterization.Post-surgical infections are the third most frequent nosocomialinfection and account for 17% of all nosocomial infections reported tothe National Nosocomial Infection Surveillance System (NNIS). The mostfrequent source of infection can be the patient's endogenous flora.Staphylococcus aureus, coagulase-negative staphylococci, andEnterococcus spp. are the pathogens most frequently isolated from SSTIs.

Symptoms of SSTI infections can include erythema, tenderness or pain,heat or localized warmth, drainage or discharge, swelling or induration,redness, or fluctuance. Patients that may benefit from treatment withthe methods of the invention include those with deep or complicatedinfections or infections that require surgical intervention, or patientswith underlying diabetes mellitus or peripheral vascular disease. Theseinfections are often caused by Gram-positive bacteria such asStaphylococcus or Streptococcus species, such as Staphylococcus aureusor Streptococcus pyogenes. Methods for treatment of a skin or softtissue bacterial infection include administering a therapeuticallyeffective amount of a N¹⁵,N¹⁵-dialkyl antibiotic compound or compositionto an individual in need of treatment, in an amount and according to adosing regime as discussed above. In one embodiment, the N¹⁵,N¹⁵-dialkylantibiotic compound is according to formula (V). In one embodiment, aN¹⁵,N¹⁵-dialkyl antibiotic compound or composition is administeredintravenously in two doses, about 5 to about 10 days apart, or about 1week apart. In some embodiments, the first dosage includes at leasttwice as much of the a N¹⁵,N¹⁵-dialkyl antibiotic compound orcomposition as the second dosage. In one embodiment, the first dosage isabout 1000 mg and the second dosage is about 500 mg.

As is understood by those skilled in the art, the dosing methodsdescribed herein can vary depending on, inter alia, the compound, thedisease and its severity, and the age, weight, etc., of the subject tobe treated, but are ascertainable by a physician without undueexperimentation.

The invention also encompasses methods for prophylactic prevention ofthe onset of a bacterial infection, for example an infection caused byStaphylococcus aureus, or by a Neisseria or Clostridium genus bacterium.In a prophylactic method of the invention, a prophylactically effectiveamount of a N¹⁵,N¹⁵-dialkyl antibiotic compound or composition isadministered to an individual who may be susceptible to contracting abacterial infection, for example, through a medical procedure. TheN¹⁵,N¹⁵-dialkyl antibiotic compound or composition can be administeredin an amount sufficient to provide a prophylactically effective plasmalevel for at least about 1 day, at least about 3 days, at least about 5days, or at least about one week or longer. The N¹⁵,N¹⁵-dialkylantibiotic compound or composition can be administered, for example,parenterally, e.g., via i.m., i.v., i.p., s.c., or i.t. injection, priorto, subsequent to, or at the same time as surgery as a preventative stepagainst infection. The N¹⁵,N¹⁵-dialkyl antibiotic compound orcomposition can be administered immediately prior or subsequently to,one or more days or about one week prior or subsequently to, or duringan invasive medical procedure such as surgery or a stay in a medicalcare facility such as a hospital to prevent infection. A prophylacticmethod can be used in any situation in which it can be possible orlikely that an individual may contract a bacterial infection, includingsituations in which an individual has been exposed to or can be likelyto be exposed to a bacterially infected individual. For prophylacticmethods, N¹⁵,N¹⁵-dialkyl antibiotic compound or composition can beadministered as either a single dose or as two or more doses of equal ordifferent amount that are administered several days to about one weekapart. In one embodiment, a N¹⁵,N¹⁵-dialkyl antibiotic compound orcomposition can be administered prior to or simultaneously withinsertion of an intravenous catheter in order to prevent a bloodstreamrelated infection. In one embodiment, the N¹⁵,N¹⁵-dialkyl antibioticcompound is aaccording to formula (V).

For prophylactic methods, a N¹⁵,N¹⁵-dialkyl antibiotic compound orcomposition can be administered in a single dose or in multiple doses,according to any of the dosing schemes described above. AN¹⁵,N¹⁵-dialkyl antibiotic compound or composition can be administeredas a single dose comprising about 0.1 to about 3 grams, or about 0.1 toabout 1 gram, e.g., about 0.25 gram or about 0.5 gram. In oneembodiment, a single dose of about 0.25 gram can be administeredintravenously over a time frame of about 2 minutes to about 1 hour,e.g., about 30 minutes. In another embodiment, the N¹⁵,N¹⁵-dialkylantibiotic compound or composition can be administered intravenouslysimultaneously with administration of another pharmaceutical (e.g.,antibiotic) treatment.

In any of the therapeutic or prophylactic methods described above, theN¹⁵,N¹⁵-dialkyl antibiotic compound or composition can be administeredeither simultaneously or sequentially with at least one otherantibiotic. In some embodiments, at least one other antibiotic that canbe effective (e.g., bactericidal) against one or more Gram-negativebacterial species and/or a Gram-positive bacterial strain against whichthe N¹⁵,N¹⁵-dialkyl antibiotic compound is not effective can beadministered in addition to the N¹⁵,N¹⁵-dialkyl antibiotic. In someembodiments, N¹⁵,N¹⁵-dialkyl antibiotic compound and at least oneantibiotic that can be effective (e.g., bactericidal) against at leastone Gram-negative bacterial species can be administered as a mixture inthe dalbavancin composition.

Pharmaceutical Compositions

The invention provides pharmaceutical compositions formulated foradministration of a N¹⁵,N¹⁵-dialkyl antibiotic compound or compositionaccording to the methods described above. Pharmaceutical compositions ofthe invention can be in the form of a unit dose of a N¹⁵,N¹⁵-dialkylantibiotic compound or composition that includes an amount of aN¹⁵,N¹⁵-dialkyl antibiotic compound or composition sufficient to providea therapeutically or prophylactically effective plasma level of theN¹⁵,N¹⁵-dialkyl antibiotic compound or composition for several days, atleast about 3 days, at least about 5 days, or at least about one week orlonger when the compound or composition is administered to anindividual, and a pharmaceutically acceptable carrier. A therapeuticallyor prophylactically effective plasma level of N¹⁵,N¹⁵-dialkyl antibioticcompound or composition can be at least about 4 mg per liter of plasma.Plasma levels of N¹⁵,N¹⁵-dialkyl antibiotic compound or composition canbe measured by methods that are well known in the art, such as liquidchromatography, mass spectrometry, or microbiological bioassay. As isknown by those skilled in the art, levels of other N¹⁵,N¹⁵-dialkylantibiotic compounds or compositions in the serum can be quantitated inthe serum using similar methodologies.

N¹⁵,N¹⁵-dialkyl antibiotic compounds and compositions, can optionally bein a pharmaceutically acceptable form for administration to anindividual, for example, as a pharmaceutically acceptable, non-toxicsalt.

Examples of suitable salts of a N¹⁵,N¹⁵-dialkyl antibiotic compoundinclude salts formed by standard reaction with both organic andinorganic acids such as, for example, hydrochloric, hydrobromic,sulfuric, phosphoric, acetic, trifluoroacetic, trichloroacetic,succinic, citric, ascorbic, lactic, maleic, glutamic, camphoric,glutaric, glycolic, phthalic, tartaric, lauric, stearic, salicylic,methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic, andthe like acids. Representative examples of bases that can form saltswith dalbavancin include alkali metal or alkaline earth metal hydroxidessuch as sodium, potassium, calcium, magnesium, and barium hydroxide,ammonia and aliphatic, alicyclic, or aromatic organic amines such asmethylamine, dimethylamine, diethylamine, ethanolamine, and picoline.(See, for example, U.S. Pat. No. 5,606,036.)

In some embodiments, a pharmaceutically acceptable aqueous formulationof a N¹⁵,N¹⁵-dialkyl antibiotic compound or composition can be providedthat is suitable for parenteral administration, such as, for example,intravenous injection. In one embodiment, the N¹⁵,N¹⁵-dialkyl antibioticcompound is according to formula (V). For preparing such an aqueousformulation of a N¹⁵,N¹⁵-dialkyl antibiotic compound or composition,methods well known in the art may be used, and any pharmaceuticallyacceptable carriers, diluents, excipients, or other additives normallyused in the art may be used. In one embodiment, a pharmaceuticallyacceptable aqueous formulation for intravenous injection includes 5%dextrose.

A pharmaceutical composition for parenteral administration comprises aN¹⁵,N¹⁵-dialkyl antibiotic compound or composition and a physiologicallyacceptable diluent such as deionized water, physiological saline, 5%dextrose, water miscible solvent (e.g., ethyl alcohol, polyethyleneglycol, propylene glycol, etc.), non-aqueous vehicle (e.g., oil such ascorn oil, cottonseed oil, peanut oil, and sesame oil), or other commonlyused diluent. The formulation may further comprise a solubilizing agentsuch as polyethylene glycol, polypropylene glycol, or other knownsolubilizing agent, buffers for stabilizing the solution (e.g.,citrates, acetates, and phosphates) and/or antioxidants (e.g., ascorbicacid or sodium bisulfite) (See, e.g., U.S. Pat. No. 6,143,739). Othersuitable pharmaceutical carriers and their formulations are described in“Remington's Pharmaceutical Sciences” by E. W. Martin. As is known inthe art, pharmaceutical preparations of the invention can also beprepared to contain acceptable levels of particulates (e.g.,particle-free) and to be non-pyrogenic (e.g., meeting the requirementsof an injectable in the U.S. Pharmacopeia).

In one embodiment, a pharmaceutical composition is provided bydissolving a dried (e.g., lyophilized) dose of a N¹⁵,N¹⁵-dialkylantibiotic compound or composition, often comprising a stabilizer ormixture of stabilizers, in an amount of water and preferably deionizedwater in a volume sufficient for solubilization. For example, the amountof water sufficient for solubilization can be approximately 10 mL andthe resulting pH can be above 3.0, and about 3.5 to 4.5. Diluting thissolution by adding it to a second amount of an aqueous diluent,containing 5% dextrose, such as an amount contained in a drip bag forintravenous administration, can raise the pH of the solution to about 5to 5.5. In another embodiment, the pH of the solution in a drip bag canbe about 4.5. The second amount of aqueous solution can be deionized orsterile, or both deionized and sterile. In one embodiment, the aqueousdiluent is 5% dextrose. Other solubilization methods, and theN¹⁵,N¹⁵-dialkyl antibiotic formulations thereof, will be readilyapparent to those skilled in the art.

Pharmaceutical compositions for parenteral administration can be made upin sterile vials containing one or more unit doses of theN¹⁵,N¹⁵-dialkyl antibiotic compound or composition in a therapeuticallyor prophylactically effective amount as described above, optionallyincluding an excipient, under conditions in which bactericidaleffectiveness of N¹⁵,N¹⁵-dialkyl antibiotic compound or composition canbe retained. The compound or composition may or may not be in the formof a dry (e.g., lyophilized) powder. Prior to use, a physiologicallyacceptable diluent can be added and the solution withdrawn via syringefor administration to a patient. A pharmaceutical formulation asdescribed above can be sterilized by any acceptable means including, forexample, e-beam or gamma sterilization methods, or by sterilefiltration.

A formulation for parenteral administration can include theN¹⁵,N¹⁵-dialkyl antibiotic compound or composition at a concentrationsuch as about 0.1 to about 100 mg, about 0.5 to about 50 mg, about 1 toabout 10 mg, about 1 to about 5 mg, or about 2 to about 4 mg of theN¹⁵,N¹⁵-dialkyl antibiotic compound or composition per ml of finalpreparation. In one embodiment, the N¹⁵,N¹⁵-dialkyl antibiotic compoundis according to formula (V).

In some embodiments, a pharmaceutical composition in accordance with theinvention comprises a mixture of a N¹⁵,N¹⁵-dialkyl antibiotic compoundor composition and one or more additional antibiotics. Preferably, atleast one non-dalbavancin antibiotic in the mixture can be effective(e.g., bactericidal) against one or more species of Gram-negativebacteria, such as, for example, azthreonam, and/or against one or moreGram-positive bacterial strains against which the N¹⁵,N¹⁵-dialkylantibiotic compound or composition can be not effective, such as, forexample, ilnezolide or daptomycin. The mixture can also include apharmaceutically acceptable carrier as described above. In someembodiments, the pharmaceutical composition comprises a N¹⁵,N¹⁵-dialkylantibiotic compound or composition and one or more additionalantibiotics. In some embodiments, the N¹⁵,N¹⁵-dialkyl antibioticcomposition comprises a N¹⁵,N¹⁵-dimethyl antibiotic compound accordingto formula (V).

In some embodiments, pharmaceutical compositions of the inventioncomprise one or more stabilizing substances that inhibit degradation ofone or more N¹⁵,N¹⁵-dialkyl antibiotic compounds to less active orinactive materials. As used herein, “stabilizing substance” or“stabilizer” refers to a substance that stabilizes the level of one ormore N¹⁵,N¹⁵-dialkyl antibiotic compounds present in the composition. A“stabilizing effective amount” refers to an amount of a stabilizersufficient to enhance long-term stability of one or more N¹⁵,N¹⁵-dialkylantibiotic compounds that can be present in the composition that can bepresent in the composition. In some embodiments, a stabilizing effectiveamount may be provided by a mixture of two or more stabilizingsubstances, each of which alone can be not present in an amountsufficient to provide a stabilizing effect.

Examples of stabilizers include, for example, nonionic substances suchas sugars, e.g., mono-, di-, or polysaccharides, or derivatives thereof,sugar alcohols, or polyols. Such stabilizing substances include, forexample, mannitol, lactose, sucrose, sorbitol, glycerol, cellulose,trehalose, maltose, raffinose, or mixtures thereof.

In one embodiment, a N¹⁵,N¹⁵-dialkyl antibiotic formulation comprisesmannitol. In another embodiment, a N¹⁵,N¹⁵-dialkyl antibioticformulation further comprises lactose. In one embodiment, theN¹⁵,N¹⁵-dialkyl antibiotic composition is formulated with a 1:2 weightratio of mannitol: N¹⁵,N¹⁵-dialkyl antibiotic compound. In anotherembodiment, the N¹⁵,N¹⁵-dialkyl antibiotic composition is formulatedwith a 1:1:4 weight ratio of mannitol:lactose: N¹⁵,N¹⁵-dialkylantibiotic compound. A combination of mannitol and lactose can provide agreater stabilizing effect than either substance alone. The pH of apharmaceutical composition of the invention can be, for example, about 2to about 9, alternatively from about 3 to about 8, alternatively fromabout 4 to about 7, alternatively from about 5 to about 6, alternativelyfrom about 3.5 to about 4.5. The pH of a pharmaceutical composition ofthe invention can also be, for example, less than about 9, alternativelyless than about 8, alternatively less than about 7, alternatively lessthan about 6, alternatively less than about 5, or alternatively lessthan about 4. The pH of a pharmaceutical composition of the inventioncan also be, for example, greater than about 2, greater than about 3,alternatively greater than about 3.5, alternatively greater than about4, alternatively greater than about 4.5, alternatively greater thanabout 5.0, alternatively greater than about 5.5, alternatively greaterthan about 6.0, alternatively greater than about 6.5, or alternativelygreater than about 7.0.

In some embodiments, one or more procedures can be employed to reduceformation of MAG (a derivative of a N¹⁵,N¹⁵-dialkyl antibiotic compoundof the invention or a dalbavancin compound lacking an acylglucoronaminemoiety) and/or other degradants. For example, freeze drying of aN¹⁵,N¹⁵-dialkyl antibiotic compound or composition in the presence of astabilizing substance, such as mannitol, can be employed to reduce theamount of MAG formed.

Storage of N¹⁵,N¹⁵-dialkyl antibiotic compounds and compositions can beat lower than ambient temperature, such as at about 5° C., to enhancestability.

Weekly dosing of a N¹⁵,N¹⁵-dialkyl antibiotic compound or composition athigh dosage levels (i.e., resulting in surprising high and long-lastingserum levels) can show a surprisingly good safety profile, similar to,or better than, that observed with the standard therapy of lower dosesof conventional antibiotics administered daily or even 2-4 times daily,as demonstrated by the Examples herein. A high dosage (i.e., resultingin high and long-lasting serum levels, e.g. 200-5000 mg) of aN¹⁵,N¹⁵-dialkyl antibiotic compound or composition can be administered,with less frequency than other antibiotics, and without adverse sideeffects, enabling improved efficacy and patient compliance.

In certain embodiments, treatment with a N¹⁵,N¹⁵-dialkyl antibioticcomposition results in a low incidence of adverse events. Seriousadverse events include any adverse drug experience occurring at any dosethat results in death, can be life-threatening, results inhospitalization or prolongation of existing hospitalization, orpersistent or significant disability or incapacity.

Kits

The invention also encompasses kits for use in methods of treatment orprophylaxis of bacterial infections. The kits can include apharmaceutical compound or composition of the invention, for examplecomprising at least one unit dose of a N¹⁵,N¹⁵-dialkyl antibioticcompound or composition, and instructions providing information to ahealth care provider regarding usage for treating or preventing abacterial infection. Instructions may be provided in printed form or inthe form of an electronic medium such as a floppy disc, CD, or DVD, orin the form of a website address where such instructions may beobtained. A unit dose of a N¹⁵,N¹⁵-dialkyl antibiotic compound orcomposition can include a dosage such that when administered to anindividual, a therapeutically or prophylactically effective plasma levelof a N¹⁵,N¹⁵-dialkyl antibiotic compound or composition can bemaintained in the individual for at least 5 days. In some embodiments, akit includes two unit dosages to be administered at least 5 days apart,about one week apart, or including a first dosage of a N¹⁵,N¹⁵-dialkylantibiotic compound or composition that can be about 1.5 to about 3times higher than the second dosage. In some embodiments, aN¹⁵,N¹⁵-dialkyl antibiotic compound or composition can be included as asterile aqueous pharmaceutical composition or dry powder (e.g.,lyophilized) composition. In one embodiment, the N¹⁵,N¹⁵-dialkylantibiotic compound is according to formula (V).

In some embodiments, suitable packaging is provided. As used herein,“packaging” refers to a solid matrix or material customarily used in asystem and capable of holding within fixed limits a N¹⁵,N¹⁵-dialkylantibiotic compound or composition suitable for administration to anindividual. Such materials include glass and plastic (e.g.,polyethylene, polypropylene, and polycarbonate) bottles, vials, paper,plastic, and plastic-foil laminated envelopes and the like. If e-beamsterilization techniques are employed, the packaging should havesufficiently low density to permit sterilization of the contents.

Kits can also optionally comprise equipment for administration of the aN¹⁵,N¹⁵-dialkyl antibiotic compound or composition, such as, forexample, syringes or equipment for intravenous administration, and/or asterile solution, e.g., a diluent such as 5% dextrose, for preparing adry powder (e.g., lyophilized) composition for administration.

Kits of the invention may also comprise in addition to the aN¹⁵,N¹⁵-dialkyl antibiotic compound or composition, a non-dalbavancinantibiotic or mixture of non-dalbavancin antibiotics, for use with the aN¹⁵,N¹⁵-dialkyl antibiotic compound or composition as described in themethods above.

The following synthetic and biological examples are offered toillustrate this invention and are not to be construed in any way aslimiting the scope of this invention.

EXAMPLES Example 15 Purification of N¹⁵,N¹⁵-Dimethyl Dalbavancin B₀

The preparation of A-40926 and subsequent synthesis of dalbavancin isdescribed above. The instant example describes the purification of theN¹⁵,N¹⁵-dimethyl antibiotic compound according to formula (V), i.e.N¹⁵,N¹⁵-dimethyl dalbavancin B₀, from a mixture of dalbavancin compoundsprepared according to the invention. As will be apparent to those ofskill in the art, N¹⁵,N¹⁵-dimethyl dalbavancin B₀ is a modifieddalbavancin B₀ with two methyl groups on N¹⁵. For the purposes of thisexample and following examples, N¹⁵,N¹⁵-dimethyl antibiotic B₀ isreferred to as “Compound A” in the sections where the compound has yetto be fully characterized. Also, for convenience in later sections,N¹⁵,N¹⁵-dimethyl dalbavancin B₀ is referred to as dalbavancin B₂.N¹⁵,N¹⁵-dimethyl antibiotic B₀, dalbavancin B₂ and Compound A are thesame molecule.

Sample Preparation

About 2 L of organic-aqueous solution from a purification of dalbavancinprepared according to the methods herein were concentrated byacetonitrile and water evaporation under vacuum. Adding diethyl ether tothe residual afforded 6 g of crude solid. After dissolution in a mixturewater/acetonitrile 90/10 v/v the solid was purified on silanized silicagel (bed volume 1 L; column id=5 cm) using a water (pH 3 aceticacid)/acetonitrile step-gradient elution. The compound A containingfractions were pooled, concentrated to small volume (few ml) andsubmitted to preparative HPLC purification.

The enriched fractions were collected. After acetonitrile evaporationunder vacuum and lyophilization a few μg of compound A were recovered.

Example 16 Identification of the N¹⁵ Amine Substituents of Compound A

Compound A has a molecular weight of 1828 Da, 14 units more thandalbavancin components B₀ and B₁ (M.W. 1814). HPLC-ESI-MS analysisdemonstrated that unlike the other components of the complex, thevariation is on the terminal amino group at N¹⁵.

Other dalbavancin compounds, possessing a methylamine group (R—NHCH₃)show in their fragmentation mass spectra (ESI-MS/MS) a neutral loss of31 units (—NH₂CH₃). The fragmentation spectrum of Compound A shows aloss of 45 units instead of 31 units. This type of neutral loss can beexplained by two structural hypotheses: N¹⁵ could be a N,N-dimethylaminegroup (R—N(CH₃)₂, tertiary amine) or a N-ethylamine group (R—NHCH₂CH₃,secondary amine).

HPLC-ESI-MS analysis of the hydrolyzed and derivatized Compound A candiscriminate between those two hypotheses. FIG. 29 shows the threepeptidic fragments originated from B₀ after acid hydrolysis. Twodi-peptides, “AA1+AA3”, with amino group N¹⁵ and a chlorine atom,“AA5+AA7”, and a tri-peptide “AA2+AA4+AA6”, with a chlorine atom, areformed. Other hydrolysis products are the fatty acid and the DMEPA4(dimethylamino-propylamino) chains.

In the case of Compound A the dipeptide “AA1+AA3” must be different asreported in FIG. 30. The correct hypothesis can be verified by reactingthe hydrolysate with a derivatizing agent that reacts with primary andsecondary amines only (see FIG. 31).

The di-peptide “AA1+AA3” of component B₀ should react with two groups ofderivatizing agent (see FIG. 31 a). In the case of Compound A twodifferent derivatized peptides “AA1+AA3” can be obtained depending onthe amino group N¹⁵ (FIG. 31 b). The two molecules, theoreticallyexpected according to the hypotheses proposed, will have molecularweights easily distinguishable by mass spectrometric analysis.

Materials

Compound A

Dalbavancin

Hydrochloric acid 37%, reagent grade, Rudi Pont cod. 750-11

Methanol HPLC grade, J. T. Backer cod. 8402

AccQ Tag™ Chemistry Package, Waters cod. WAT052880

Hydrolysis

The hydrolysis of both the sample and the standard was carried out in aPICO-TAG Work Station (Waters, Mildoford, Mass., USA).

All of the 360 μg of Compound A and about 1 mg of the dalbavancinstandard were hydrolyzed at 105° C. in the presence of 6N HCl containing1% (w/v) phenol for 24 hours. The reaction mixtures were cooled,evaporated to dryness and brought to a final volume of 500 L withmethanol.

Derivatization

Waters AccQ-Fluor™ reagent Kit was chosen for peptide derivatization.AccQ-Fluor reagent is one of the N-hydroxysuccinimide-activatedheterocyclic carbamate aminederivatizing compounds. The structure of thederivatization agent and the scheme of reaction is reported in Scheme 2.

1 mL of “Reagent Diluent” was added to the vial containing “ReagentPowder”. The vial was vortexed for 10 seconds and warmed up at 55° C.until complete dissolution. Reconstituted AccQ-Fluor reagent wasapproximately 10 mM in acetonitrile. 20 μl of hydrolyzed sample orstandard were put into a vial with conical insert and 20 μl of“AccQ-Fluor Borate Buffer” were added and vortexed briefly. At thispoint 40 μl of reconstituted AccQ-Fluor reagent were added and thesample vortexed for 30 sec. Vials were heated at 55° C. for 10 min. Inthese conditions the amino groups should react and eventual by-productsshould be minimized. After this reaction the samples were ready for HPLCanalysis.

HPLC-UV-MS Analysis

Thermo Finnigan Surveyor MS pump, diode array detector and autosampler,

ThermoQuest Finnigan LCQ Deca mass detector equipped with ESI interface.

Chromatography: Column: AccQ-Tag™ (Waters C18 NovoPak 4 _m 3.9×150 mm);Column temperature: 37° C.; Flow: 1 mL/min; Phase A: ammonium acetate140 mM pH 5 (acetic acid); Phase B: water/acetonitrile 60/40 v/v; UVdetection: 254 nm; Injection volume: 20 μL. The eluate from the columnwas split to allow simultaneous UV and mass detection.

Mass spectrometry

Sample inlet condition: capillary temperature: 200° C.

sheath gas: N₂, 40 (arbitrary units)

auxiliary gas N₂, 20 (arbitrary units)

Sample inlet voltage setting: polarity: positive

source voltage: 4.7 kV

capillary voltage: 10 V

tube lens offset: 40 V

Scan conditions: scan mode: full ms

scan range: 100-700 amu

number of microscans 3

maximum ion time 50 ms

Results

Derivatized and hydrolyzed fragments of dalbavancin were separated byHPLC and evaluated by ESI-MS (data not shown). Fragment AA1+AA3 had asize of 737 (m/z), fragment AA5+AA7 had a size of 689 (m/z), andfragment AA2+AA4+AA6 had a size of 1086 (m/z).

Derivatized and hydrolyzed fragments of Compound A were separated byHPLC and evaluated by ESI-MS. Fragment AA1+AA3 had a size of 737 (m/z),fragment AA5+AA7 had a size of 581 (m/z), and fragment AA2+AA4+AA6 had asize of 1086 (m/z).

The size of the AA5+AA7 confirmed that Compound A is an N¹⁵,N¹⁵-dimethyl compound.

Example 17 Methylation of Dalbavancin B₀

Dalbavancin B₀ was prepared following the methods outlined generallyabove and purified by HPLC following methods described in U.S. PatentApplication Publication No. 2004/0142883. A sample of dalbavancin B₀ wassubmitted to selective secondary amine N-methylation to yieldN¹⁵,N¹⁵-dimethyl antibiotic B₀. The reaction mixture was then analyzedby HPLC-UV and HPLC-UV-MS in the Example below.

Materials

Dalbavancin B₀

Water, MmilliQ grade

NaBH₃CN, sodium cyanoborohydride, Fluka cod. 71435

DMF, N,N-dimethylformamide, Carlo Erba cod. 444926

HCHO, formaldehyde solution, 36.5% water solution, Reidel de Haen cod.33220.

NaHCO₃, sodium bicarbonate, reagent grade

Method

49.5 mg of dalbavancin were dissolved in 12.5 ml of water and 1.5 mL ofDMF into a round-bottom flask (pH 3.5). Three aliquots of 200 μL of werewithdrawn and diluted with 800 μL of water (t0). 76 μL of a 36% (v/V)water solution of formaldehyde and 2.5 mg of sodium bicarbonate (pH 5.8)were added. 8 mg of NaCNBH₃ were added under stirring after sodiumbicarbonate complete dissolution. Three aliquots of 200 μL of thisreaction mixture were immediately withdrawn and diluted with 800 μL ofwater and 300 μL of CH₃CN in order to get a clear solution (t0). Thereaction mixture was left at room temperature for 30 minutes. Sampleswere taken from the reaction medium after 10 and 30 minute by extracting3 aliquots of 200 μl and diluting each with 800 μl of water and 300 μlof CH₃CN (t10 and t30).

After 30 minutes the reaction was stopped by cooling down to −20° C. theflask. The reaction was monitored by HPLC-UV and HPLC/JUV/MS analysis.

Example 18 Structural Confirmation of N¹⁵,N¹⁵-Dimethyl Antibiotic B₀

This example demonstrates that Compound A and N¹⁵,N¹⁵-dimethylantibiotic B₀ from the previous example are identical.

The reaction samples t0, t0′, t10 and t30 were analyzed by HPLC-UV andcompared with the reference standard. Chromatograms of t0′, t10 and t30were very similar, demonstrating that methylation occurs immediately.

In FIG. 33, t0′ vs t0 chromatograms are shown. Methylated B₀ showed thesame retention time as Compound A, from Example 15, above. Thehypothesis that Compound A is the methylated derivative of B₀ andconsequently that the fatty acid chain of Compound A is a 10-methylundecanoic acid was thus demonstrated.

The HPLC-UV-MS results confirmed the HPLC-UV structural conclusionreported in the last paragraph. N,N′-dimethyl B₀ peak was perfectlyoverlapped to that of Compound A.

It was thus demonstrated that the group at N¹⁵ of Compound A is adimethylamine group and not an ethylamine. The elucidated structure ofcomponent Compound A is reported in FIG. 28.

The structure of compound A was further confirmed by NMR, ESI-MS and IR.NMR spectra were recorded on a Bruker AMX 600 at 313K. The sample wasdissolved in DMSO-d₆ and proton and carbon NMR assignments weredetermined by COSY-DFTP, ROESY, and HMQC spectra. A mixing time of 350msec was selected for the ROESY experiment. Mass spectra were determinedby electrospray ionization in positive mode on a Thermoquest FinniganLCQ^(deca) at 250° C. The infrared spectrum was recorded in KBron aBruker IFS 48 instrument.

NMR peak assignments are provided in FIG. 34. Table 40 reports 1Hassignments of B2 in comparison with B₀. The NMR spectra of B2 revealmany similarities with dalbavancin B₀. The fatty acid chain contains anisopropylic terminal group. Most of the proton and carbon chemicalshifts are about the same as found for the B₀ component. The majorchemical shift deviations are observable for signals belonging to theamino acid 1. In particular, the singlet at 2.31 ppm (¹³Cδ 40.06 ppm)gives integral corresponding to six protons and NOE correlations withthe protons x1, 1f and 1e. Its chemical shift and the dipolarcorrelations suggest that this signal is due to dimethylamino groupbelonging to the first amino acid spin system. TABLE 40 B₂ B₀ δ ¹H δ ¹Hδ ¹³C ¹³C w1 — n.a. — CH₃ 2.31 2.45 30.99 x1 4.43 4.33 66.07 1b 6.706.71 118.5 1e 6.93 7.05 117.6 1f 7.00 7.23 126.65 w2 7.36 8.2  — x2 4.834.79 55.9 z2 2.81; 3.32 2.95; 3.37 n.a. 2b 7.09 7.1  130.6 2c 7.17 7.07123.4 2e 6.97 6.97 122.33 2f 7.63 7.87 131.29 w3 7.58 7.54 — x3 6.106.09 54.31 3d 6.72 6.55 107.5 3f 6.49 6.78 107.1 w4 7.64 7.44 — x4 5.605.59 54.63 4b 5.80 5.78 108.8 4f 5.11 5.08 103.8 w5 8.36 8.48 — x5 4.414.35 57.72 5b 7.10 7.13 135.01 5e 6.71 6.71 116.0 5f 6.71 6.71 125.8 w66.62 6.62 — x6 4.14 4.19 61.97 z6 5.20 5.38 70.8 6b 7.69 7.75 126.6 6e7.26 7.28 123.2 6f 7.42 7.44 126.9 w7 8.41 8.47 — x7 4.42 4.45 53.3 7d6.73 6.75 101.1 7f 6.43 6.40 107.9 AG1 5.48 5.40 101.9 AG2 3.72 3.7155.98 AG3 3.62 3.62 76.11 AG4 3.41 3.51 n.a AG5 n.a. 4.27 66.28 AG-NH7.71 7.77 — FA2 2.02 2.01 35.8 UA3 1.43 1.42 24.84 FA4-FA9 1.11-1.221.0-1.2 28.8-29.3 FA10 1.50 1.49 27.2 FA11 0.84 0.84 22.34 FA12 0.840.84 22.34 wNN 7.96 8.19 — NNa 3.31; 3.20 3.17; 3.40 35.38 NNb 1.65 1.8723.37 NNc 2.39 2.96 53.94 NNd 2.24 2.71 42.05 M1 5.27 5.32 96.7 M2 3.253.3  70.24 M3 n.a. 3.49 73.6 M4 n.a. 3.69 72.91 M5 n.a. 3.50 n.a. M6n.a. 3.50 60.68

An infrared spectrum is provided at FIG. 35. ESI-MS is provided at FIG.36. The data confirmed the structure of compound A as dalbavancin B₂(N¹⁵,N¹⁵-dimethyl dalbavancin B₀).

Example 19 Activity of Dalbavancin B₂ (N¹⁵,N¹⁵-Dimethyl Dalbavancin B₀)

N¹⁵,N¹⁵-dimethyl dalbavancin B₀, several N¹⁵-monomethyl dalbavancincompounds and several known antibiotics were isolated and tested invitro for their antibacterial activity. The compounds were preparedaccording to the examples above and the methods described in U.S. PatentApplication Publication No. 2004/0142883. Individual dalbavancincompounds were purified by HPLC.

Sample Preparation

Isolated N¹⁵-monomethyl dalbavancin compounds and N¹⁵,N¹⁵-dimethylantibiotic compounds were dissolved in 0.01N HCl:DMSO: 95:5 v/v. Beforedissolution, each sample was analyzed by HPLC and quantified. Thesolutions were prepared at 256 μg/ml for the N¹⁵-monomethyl dalbavancincompounds (A₀, A₁, B₀, and B₁ dalbavancin) and at 128 μg/ml for theisolated N¹⁵,N¹⁵-dimethyl antibiotic B₀ (referred to as “B₂ dalbavancin”in the tables below).

The chromatogramic purity of the principal peak was evaluated in area %vs. total chromatogram area as reported in Table 41: TABLE 41CHROMATOGRAPHIC PURITY OF PRINCIPAL PEAK OF VARIOUS N¹⁵-MONOMETHYL ANDN¹⁵,N¹⁵-DIMETHYL ANTIBIOTIC COMPOUNDS Concentration ID Status (μg/ml) %Main Peak A₀ dalbavancin Solution 256 82 A₁ dalbavancin Solution 256 80B₀ dalbavancin Solution 256 96 B₁ dalbavancin Solution 256 79 B₂dalbavancin Solution 128 72

Microbiological Characterization

The compounds tested included the isolated N¹⁵-monomethyl dalbavancincompounds and N¹⁵, N¹⁵-dimethyl antibiotic compounds described above aswell as a dalbavancin composition (“DA 025/A”) comprising aN¹⁵,N¹⁵-dimethyl antibiotic compound, vacomycin (VA) (Sigma ChemicalCo., St. Louis, Mo.), Gentamycin (GE) (Sigma Chemical Co., St. Louis,Mo.), Penicillin G (Pen.G) (Sigma Chemical Co., St. Louis, Mo.), andAmphotericin B (Amph.B) (Sigma Chemical Co., St. Louis, Mo.).

Microorganisms

The microorganisms used were reference strains obtained from theAmerican Type Culture Collection (ATCC) (Rockville, Md.), SmithKline andFrench Laboratories (SKF), and the Upjohn Company (UC) (Kalamazoo,Mich.).

Minimum Inhibitory Concentration (MIC) Determinations

MICs were determined by the broth microdilution methodology followingthe standard NCCLS procedure (NCCLS Document M7-A6, Vol. 23, No. 2,“Methods for dilution antimicrobial susceptibility tests for bacteriathat grow aerobically.” Approved Guideline. January 2003, which can beherein incorporated by reference in its entirety), with or without 30%adult bovine serum (BS) (PAA Laboratories GMBH, Haidmannweg PaschingAustria), using bacterial innocula at approximately 5×10⁵ CFU/ml. Themedia employed included cation-adjusted Müller Hinton broth (DifcoLaboratories, Detroit, Mich.) that was used that was adjusted with CaCl₂and MgCl₂ to a final concentration of 20 mg/L and 10 mg/L, respectively.Tests were read following 20-24 hours of incubation at 35° C.

Results

The MIC results are summarized in Table 42 below. Against a panel ofgram positive bacteria, the activity of the various isolatedN¹⁵-monomethyl dalbavancin compounds and the N¹⁵,N¹⁵-dimethyl antibioticcompound (“B₂”) was comparable or slightly higher than the DAcomposition, which comprises a mixture of the N¹⁵,N¹⁵-dialkyl antibioticcompounds. Blank solution (HCL 0.01N/DMSO 95:5) used to dissolve testcompounds was inactive.

Significantly, the N¹⁵,N¹⁵-dimethyl antibiotic compound (“B₂”) showedactivity against the gram-positive bacteria that was comparable to orgreater than the N¹⁵-monomethyl dalbavancin compounds. For instance, theactivity of the N¹⁵,N¹⁵-dimethyl antibiotic compound (“B₂”) iscomparable to or greater than the activity of B₀ across the range ofgram-positive bacteria.

Quite significantly, the N¹⁵,N¹⁵-dimethyl antibiotic compound (“B₂”)also showed activity against the range of gram-negative bacteria tested.In fact, the only compound that was active against the representativepanel of gram-negative bacteria in this assay was the isolatedN¹⁵,N¹⁵-dimethyl antibiotic compound (“B₂”), which showed activity witha MIC range of 16-32 mg/L.

The MIC values of the VA and GE reference compounds against the ATCCreference strains were in the range of NCCLS values. TABLE 42 IN VITROACTIVITY OF VARIOUS N¹⁵,N¹⁵-DIALKYL ANTIBIOTIC COMPOUNDS AND REFERENCESTRAINS MIC (mg/L) DA Microorganism A₀ A₁ B₀ B₁ B₂ 025/A VA GE Pen.GAmph.B Blank* Staphylococcus 0.125 0.125 0.25 0.125 0.125 0.25 1 0.50.06 >64 >1:2 aureus Smith ATCC 19636 S. aureus Smith + 30% 1 2 2 2 2 22 ≦0.125 0.06 >64 >1:2 BS S. aureus ATCC 0.125 0.125 0.125 0.125 0.1250.25 2 8 8 >64 >1:2 29213 ref. strain¹ S. epidermidis 0.125 0.125 ≦0.06≦0.06 ≦0.03 0.125 2 ≦0.125 >32 >64 >1:2 ATCC 12228 Streptococcus 0.015≦0.06 ≦0.06 ≦0.06 ≦0.03 0.06 1 4 ≦0.03 32 >1:2 pyrogenes SKF 13400 S.pneumoniae 0.125 ≦0.06 ≦0.06 ≦0.06 ≦0.03 0.015 0.5 8 ≦0.03 >64 >1:2Felton UC41 Enterococcus 0.125 0.125 0.125 0.125 0.06 0.25 1 832 >64 >1:2 faecalis ATCC 7080 E. faecalis ≦0.06 0.125 0.125 0.125 0.1250.25 2 32 4 >64 >1:2 ATCC29212 ref. strain² Bacillus subtilis ≦0.06≦0.06 ≦0.06 ≦0.06 ≦0.03 ≦0.007 ≦0.125 0.25 ≦0.03 >64 >1:2 ATCC 6633Escherichia coli >64 >64 >64 >64 32 >64 >128 2 >32 >64 >1:2 SKF 12140 E.coli ATCC25922 >64 >64 >64 >64 32 >64 >128 1 >32 >64 >1:2 ref. strain³Proteus vulgaris >64 >64 >64 >64 32 >64 >128 2 >32 >64 >1:2 ATCC 881Pseudomonas >64 >64 >64 >64 16 >64 >128 1 >32 >64 >1:2 aeruginosas ATCC10145 Candida albicans >64 >64 >64 >64 32 >64 >128 >128 >32 0.125 >1:2SKF 2270*Lowest dilution factor not showing activity.Quality control range of MICs for reference strains (NCCLS M100 S-12,Vol. 22, No. 1, Jan. 2002):¹VA 0.5-2 mg/L²VA 1-4 mg/L³GE 0.25-1 mg/L

Example 20 Preparation of a Dalbavancin Composition ComprisingDalbavancin B₂

A composition comprising purified dalbavancin B₀, dalbavancin B₁ anddalbavancin B₂ was produced by preparative HPLC.

A dalbavancin mixture prepared according to the methods above waspurified by HPLC. The purification was carried out on Kromasil C18, 16μm, 100 Å pore size. The buffer system was a 50 mmolar NH₄H₂PO₄ aqueoussolution with the pH adjusted to 5.5. The mobile phase for the elutionwas mixture 66/34 buffer/acetonitrile. The process yielded a dalbavancincomposition comprising 97% dalbavancin B₀, dalbavancin B₁ anddalbavancin B₂ by HPLC analysis. A typical analytical HPLC chromatogramis at FIG. 37, with dalbavancin Boat about 29.901, dalbavancin B₁ atabout 30.79 (unlabeled) and dalbavancin B₂ at about 33.282.

Characterization of IsoB₀

The mass spectrum of IsoB₀ (FIG. 38) shows an ion at m/z 1817corresponding to the mono-protonated molecule and other ions due tocation adducts or partial source fragmentations. The molecular weight of1816 Da and the fragmentation pattern is the same as that observed for Bcomponents, which supports the identification of IsoB₀ as a diastereomerof B₀ (the same molecule with one or more stereocenters having invertedchirality).

The level of IsoB₀ was found to increase whenever the pH increased toomuch in the various preparation steps. Therefore, IsoB₀ was prepared bybasic treatment of dalbavancin.

Sample Preparation (BI-K0096)

About 300 mg of IsoB₀ were obtained from 10 g of dalbavancin batch 027by long (approximately 165 hours) treatment with NaOH in water (pH 12.7)at room temperature. After neutralization, the reaction product wasrecovered as a brown crude solid that was subjected to a doublechromatographic purification on a silanized silica gel column performedeluting with water (pH 3.5; acetic acid)/aceotnitrile in step-gradientmode.

From the rich fractions, a solid 79.98% area of BI-K0096 was isolated.The HPLC profile is reproduced in FIG. 39.

To verify the correspondence of BI-K0096 with the dalbavancin impurityIsoB₀, the prepared compound BI-K0096 was analyzed by HPLC using threedifferent methods. In all of the conditions tested, BI-K0096 waschromatographically identical to the IsoB₀ impurity.

Structural Analysis

MS and MS/MS spectra are reported in FIGS. 40 A-C. These mass spectraare identical to that of component B₀. From this mass analysis, it isnot possible to confirm the stereochemical variation.

¹H and ¹³C NMR spectra of IsoB₀, recorded in DMSO-d⁶ on a 600 MHzspectrometer, revealed many similarities with dalbavancin's spectra butalso important differences (see FIGS. 41 and 42, respectively). Theassignments for IsoB₀ and B₀ are reported in TABLE 43 and thecorresponding location of the protons are identified in FIG. 43. TABLE43 NMR Assignments of Dalbavancin Iso B₀ iso B₀ Assignment δ ¹H δ ¹³C w1n.a. — CH₃ 2.24 33.70 x1 4.26 64.65 1b 7.13 120.5 1e 6.82 116.6 1f 6.97120.87 w2 8.19 — x2 4.53 55.67 z2 2.80 38.59 2b 7.01 n.a. 2c 6.90 n.a.2e 7.29 n.a 2f 7.22 n.a. w3 8.44 — x3 5.34 54.65 3d 6.51 104.7 3f 6.0 101.36 w4 8.59 — x4 5.38 55.07 4b 5.74 109.07 4f 5.12 104.5 w5 8.32 — x54.56 53.20 5b 7.16 135.3 5e 6.70 115.88 5f 6.70 n.a. w6 6.59 — x6 4.2162.04 z6 5.26 71.27 6b 7.77 126.8 6e n.a. n.a. 6f 7.42 126.9 w7 8.56 —x7 4.45 n.a. 7d 6.75 101.5 7f 6.43 108.18 AG1 5.23 102.0 AG2 3.72 55.91AG3 3.55 n.a. AG4 3.48 n.a. AG5 3.26 n.a. AG-NH 7.60 — FA2 2.0  n.a. FA31.41 n.a. FA4-FA9 1.11-1.30 n.a. FA10 1.49 na. FA11 0.85 n.a. FA12 0.85n.a. wNN 8.05 — NNa 3.15; 3.23 n.a NNb 1.63 n.a. NNc 2.30 n.a. NNd 2.16n.a. M1 5.21 97.7 M2 n.a. n.a. M3 n.a. n.a. M4 n.a. n.a. M5 n.a. n.a. M6n.a. n.a.

For some parts of the molecule, proton and carbon chemical shifts areabout the same as for the B₀ component. The major chemical shiftdeviations are observable for signals belonging to the amino acidssequence 1-4. The protons of these spin systems show a negligiblepositive or negative Δδ (chemical shift difference). The amino acid 3 inparticular exhibits the greater number of relevant changes (x3, w3, 3f,3d); x3 is also the only resonance displaying an important modificationof its ³J_(HH), the coupling constant CH(x3)-NH(w3) is now 6.54 Hzcompared to about 10.4 in B₀. Furthermore, ROESY experiments indicatedifferences in dipolar correlations for x3 as well as for most protonsclose to x3. The NMR findings reported and literature data published onteicoplanin epimers make clear that x3 is the epimerization center andthe epimerization induces a change in the conformation in at least in apart of the molecule.

Microbiological Characterization

The compounds used were:

-   -   IsoB₀ as previously described above    -   Dalbavancin (DA) (BI-K397 batch 025/A/AS1) Ref std.    -   Vancomycin (VA) (batch 121K1140 Sigma Chemical Co. St Louis,        Mo., USA).    -   Gentamicin (GE) (batch 57H1099 Sigma Chemical Co. St Louis, Mo.,        USA)    -   Penicillin G (Pen.G) (batch 43H1134 Sigma Chemical Co. St Louis,        Mo., USA)    -   Amphotericin B (Amph.B) (batch 61H4039 Sigma Chemical Co. St        Louis, Mo., USA).

Vancomycin, Dalbavancin and Amphotericin B were dissolved at 10 mg/mL indimethyl sulfoxide (DMSO) and diluted in distilled water; gentamycin andPenicillin G were dissolved in distilled water.

Media

Müller Hinton broth (Difco Laboratories, Detroit, Mich., USA) adjustedwith CaCl₂ and MgCl₂ to a final concentrations of 20 mg/L and 10 mg/Lrespectively.

Sera

Adult bovine serum (BS) (batch A05123-159 PAA Laboratories GmBHHaidmannweg Pasching Austria).

Microorganisms

The microorganisms used are reference standard strains from the AmericanType Culture Collection (ATCC, Rockville, USA), Smith Kline and FrenchLaboratories (SKF) and the Upjohn Company (UC, Kalamazoo, Mich., USA).

Minimum Inhibitory Concentration (MIC)

MICs were determined by the broth microdilution methodology followingthe standard NCCLS procedure [13], with or without 30% bovine serum,using bacterial inocula of approximately 5×10⁵ CFU/mL. Tests were readafter 20-24 h incubation at 35° C.

Results

Against a panel of gram-positive bacteria, the activity of isoB₀ is verysimilar to dalbavancin. (See TABLE 44) TABLE 44 In Vitro Activity ofIsoB₀ and Reference Compounds MIC (mg/L) DA Blank Microorganism IsoB₀025/A VA GE Pen.G Amph. B activity* Staphylococcus aureus Smith 0.5 0.251 0.5 0.06 >64 >1:2 ATCC19636 S. aureus Smith + 30% BS 16 2 2 ≦0.1250.06 >64 >1:2 S. aureus ATCC29213 ref. strain¹ 0.25 0.25 2 8 8 >64 >1:2S. epidermidis ATCC12228 0.125 0.125 2 ≦0.125 >32 >64 >1:2 Streptococcuspyogenes SKF13400 0.06 0.06 1 4 ≦0.03 32 >1:2 S. pneumoniae Felton UC410.125 0.015 0.5 8 ≦0.03 >64 >1:2 Enterococcus faecalis ATCC7080 0.250.25 1 8 32 >64 >1:2 E. faecalis ATCC29212 ref. strain² 0.25 0.25 2 324 >64 >1:2 Bacillus subtilis ATCC6633 ≦0.06 ≦0.007 ≦0.125 0.25≦0.03 >64 >1:2 Escherichia coli SKF12140 64 >64 >128 2 >32 >64 >1:2 E.coli ATCC25922 ref. strain³ 64 >64 >128 1 >32 >64 >1:2 Proteus vulgarisATCC881 >64 >64 >128 2 >32 >64 >1:2 Pseudomonas aeruginosas >64 >64 >1281 >32 >64 >1:2 ATCC10145 Candida albicans SKF2270 >64 >64 >128 >128 >320.125 >1:2

Although the foregoing invention has been described in some detail byway of illustration and examples for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications may be practiced without departingfrom the spirit and scope of the invention. Therefore, the descriptionshould not be construed as limiting the scope of the invention, which isdelineated by the appended claims.

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes andto the same extent as if each individual publication, patent, or patentapplication were specifically and individually indicated to be soincorporated by reference.

1-69. (canceled)
 70. A chemical compound of the formula

wherein R² is a C₁₀₋₁₄ acyl.
 71. The compound of claim 70, wherein R² isselected from the group consisting of 8-methyl nonanoic acid, n-decanoicacid, 9-methyl-decanoic acid, n-undecanoic acid, 10-methyl-undecanoicacid, n-dodecanoic acid, 11-methyl-dodecanoic acid, n-tridecanoic acid,12-methyl-tridecanoic acid, and n-tetradecanoic acid.
 72. The compoundof claim 70, wherein R² is 10-methyl-undecanoic acid.
 73. A compound ofthe formula


74. A compound according to formula 1, or a pharmaceutically acceptablesalt or solvate thereof:

wherein: M is hydrogen, α-D-mannopyrannosyl, or6-O-acetyl-α-D-mannopyrannosyl; G is hydrogen, glucuronamine oracylglucuronamine; and X is an aminoalkylamino.
 75. The compound ofclaim 74, having formula 2:

76-163. (canceled)